Diagnosis and treatment of cancer involving the notch pathway

ABSTRACT

The present invention provides methods and compositions for the diagnosis and treatment of cancer, including cancers involving the NOTCH pathway. In particular, the present invention provides methods and compositions for the diagnosis of mucoepidermoid carcinoma, the most common malignant salivary gland tumor. The present invention further provides methods and compositions for the diagnosis of other tumors associated with the t(11;19)(q14-21;12-13) translocation.

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/302,788 filed on Jul. 3, 2001, which is hereinincorporated by reference in its entirety. This invention was made inpart during work supported by Federal funds from the National CancerInstitute, and as such the government has certain rights in theinvention.

FIELD OF THE INVENTION

[0002] The present invention provides methods and compositions for thediagnosis and treatment of cancer, including cancers involving the NOTCHpathway. In particular, the present invention provides methods andcompositions for the diagnosis of mucoepidermoid carcinoma, the mostcommon malignant salivary gland tumor. The present invention furtherprovides methods and compositions for the diagnosis of other tumorsassociated with the t(11;19)(q14-21;12-13) translocation.

BACKGROUND OF THE INVENTION

[0003] The annual U.S. incidence rate of head and neck cancer isapproximately 40,000 cases (Vokes et al., New Eng. J. Med., 328:184[1993]). Although salivary gland tumors differ in their etiology,histology and standard therapy from most head and neck cancer, thesecancers represent a significant threat to human health. Salivary glandtumors arise from either one of the three major salivary glands or fromthe minor salivary glands that line the mucosa of the upperaerodigestive tract. Histologically, these tumors are veryheterogeneous, and include mucoepidermoid cancers, pleomorphic adenoma,and adenoid cystic carcinomas as the more frequent observed tumor types.Treatment of these tumors is predominantly surgical, with post-operativeradiotherapy being frequently administered. For unresectable tumors,neutron irradiation has been used in place of conventional radiotherapy.Chemotherapy is typically reserved for patients with recurrent ormetastatic disease.

[0004] Mucoepidermoid carcinoma (MEC) is the most common malignant humansalivary gland tumor which can arise from both major (parotid) and minorsalivary glands, including serous/mucous glands within the pulmonarytracheobronchial tree (Calcaterra, in Cancer Treatment, 4^(th) ed.(Haskell, ed.), W. B. Saunders Company, Philadelphia, [1995], at pages721-726). These salivary gland tumors may be deadly, due to theirtendency to grow locally and recur aggressively, if not completelyexcised. However, complete excision is difficult due to thethree-dimensional growth pattern of these tumors, which make itdifficult for the surgeon to accurately determine when clean marginshave been achieved. Pathologic analysis using light microscopy iscurrently employed to assess tumor margins and to help determine theneed for post-operative radiotherapy. However, this approach does notnecessarily provide sufficient sensitivity for optimal patientmanagement. In addition, both surgeons and patients desire minimalsurgical approaches for cosmetic reasons, as well as to preserve nervefunction to the facial area. Thus, methods and compositions suitable forthe rapid and reliable diagnosis of these and other aggressive tumorsare needed.

SUMMARY OF THE INVENTION

[0005] The present invention provides methods and compositions for thediagnosis and treatment of cancer, including cancers involving the NOTCHpathway. In particular, the present invention provides methods andcompositions for the diagnosis of mucoepidermoid carcinoma, the mostcommon malignant salivary gland tumor. The present invention furtherprovides methods and compositions for the diagnosis of other tumorsassociated with the t(11;19)(q14-21;12-13) translocation.

[0006] The present invention provides compositions and methods todefinitively diagnose mucoepidermoid carcinomas and other tumors thatare associated with the t(11;19)(q14-21;p12-13) translocation. In someembodiments, the present invention provides diagnostic means thatutilize minimal biopsy samples. In particularly preferred embodiments,the present invention provides methods and compositions suitable fortesting of fine needle aspirate samples. In some additional preferredembodiments, the present invention provides methods and compositions forFISH analysis of tumor cells, while in alternative embodiments, thepresent invention provides methods and compositions for RT-PCR analysisof RNA extracted from tumor cells.

[0007] The present invention also provides BAC clones useful forconsistent markers of the translocation of interest. In particularlypreferred embodiments, the two adjacent BAC clones from human chromosome11q21, designated as “RP11-676L3” and “RP11-16K5” are used as consistentmarkers for the translocation. RP11-676L3 contains exon 1 of the MAML2gene and is retained on the derivative 11 chromosome, while RP11-16K5contains exons 2-5 of the MAML2 gene and is translocated to thederivative 19 chromosome. FISH hybridization with these BAC probesprovides means to detect normal chromosome 11 and provides evidence forthe t(11;19) translocation.

[0008] The present invention also provides methods and compositions forRT-PCR analysis using gene-specific oligonucleotides. In someembodiments, the present invention provides means to detect specificMECT1/MAML2 fusion products in biopsy samples. These embodiments of thepresent invention provide much greater sensitivity than the conventionallight microscopy methods that are presently routinely used. In preferredembodiments, these methods and compositions provide means to obtain datawithin 24 hours. In particularly preferred embodiments, withoptimization of reagents and test system parameters, the testinganalysis is completed within 6 to 7 hours.

[0009] The present invention also provides methods and compositions forimprovement of pre- and/or post-operative management of patients withmucoepidermoid carcinomas and other tumors associated with the t(11;19)translocation. In some embodiments, the present invention provides meansfor three-dimensional mapping of the precise location of residual tumormaterial. In particularly preferred embodiments, the present inventionprovides means to map irregular tumor margins in three dimensions,either intraoperatively and/or post-operatively. Thus, the molecularmapping of tumor margin methods and compositions of the presentinvention facilitate treatment regimens, as the data obtained using thepresent invention help the surgeon and patient determine whether repeatresection is required, and/or whether post-operative radiation therapyis necessary and/or desirable. In some embodiments, FISH analysis isutilized, while in other embodiments, RT-PCR is utilized.

[0010] The present invention further provides methods and compositionsto analyze disruptions in the NOTCH signal transduction pathway. Inparticularly preferred embodiments, tumors carrying the t(11;19)translocation are identified as having disruptions in the NOTCH signaltransduction pathway. In some embodiments, the aspects of the NOTCHpathway associated with differentiation of central nervous system andhematopoietic tissues, as well as the genesis of epithelial carcinomasare involved. In some embodiments, the present invention provides meansto identify the mutant fusion product MECT1/Mastermind-like 2, whichretains the transactivation domain for the NOTCH/Mastermind complex, butlacks the amino-terminal binding site for the NOTCH product. Inalternative embodiments, the present invention provides means to analyzeinhibitors of the transactivation domain of MAML2 for their ability toact as potential targets for the treatment of mucoepidermoid carcinomasand other tumors associated with NOTCH gene deregulation. In someembodiments, the inhibitors are small molecules. In alternativeembodiments, the inhibitors are monoclonal or polyclonal antibodies.

[0011] The present invention provides methods of screening a tissuesample from a subject for a t(11;19)(q14-21;p12-13) translocation,comprising detecting the presence of a MECT1-MAML2 chimeric nucleic acidin a tissue sample. In some embodiments, the tissue sample comprisesbiopsy material. In preferred embodiments, the biopsy material comprisescells from a salivary gland tumor. In related embodiments, the salivarygland tumor is selected from the group consisting of a mucoepidermoidcancer, a pleomorphic adenoma, and a adenoid cystic carcinoma. In someembodiments, the MECT1-MAML2 chimeric nucleic acid comprises DNA. Inrelated embodiments, the detecting is by fluorescence in situhybridization, by amplifying at least a portion of said MECT1-MAML2 DNAby polymerase chain reaction, or by Southern blot. In other embodiments,the MECT1-MAML2 chimeric nucleic acid comprises RNA. In relatedembodiments, the detecting is by amplifying at least a portion of aMECT1-MAML2 mRNA by reverse-transcriptase polymerase chain reaction, byNorthern blot, or by microarray.

[0012] The present invention also provides methods of screening a tissuesample from a subject for a t(11;19)(q14-21;p12-13) translocation,comprising detecting the presence of a MECT1-MAML2 chimeric protein in atissue sample. In related embodiments, the detecting is by immunoblot,or by immunofluorescence analysis.

[0013] In other embodiments, the present invention provides kits forscreening a tissue sample from a subject for a t(11;19)(q14-21;p12-13)translocation, comprising: a reagent capable of specifically detectingthe presence of a MECT1-MAML2 chimeric nucleic acid in a tissue sample;and instructions for using the kit for screening a tissue sample from asubject for a t(11;19)(q14-21;p12-13) translocation. In someembodiments, the reagent comprises a first nucleic acid probecomplementary to at least a portion of MECT1 exons 2-18, and a secondnucleic acid probe complementary to at least a portion of MAML2 exon 1.In other embodiments, the reagent comprises a first nucleic acid probecomplementary to at least a portion of MECT1 exon 1, and a secondnucleic acid probe complementary to at least a portion of MAML2 exons2-5. In related embodiments, the reagent comprises a first bacterialartificial chromosome designated as RP11-676L3, and a second bacterialartificial chromosome designated as RP11-16K5. In some embodiments, thefirst nucleic acid probe comprises a sense oligonucleotide, and thesecond nucleic acid probe comprises an antisense oligonucleotide.

[0014] The present invention also provides methods of screeningcompounds, comprising: providing: a cell containing a MECT1-MAML1chimeric gene; and at least one test compound; and contacting the cellwith the test compound; and detecting a change in MECT1-MAML2 expressionin the cell in the presence of the test compound relative to the absenceof the test compound. In some embodiments, the cell is selected from thegroup consisting of a cell transfected with a MECT1-MAML2 expressionvector, and a cell with a t(11;19)(q14-21;p12-13) translocation. Inother embodiments, the cell is selected from the group consisting of acell in vitro and a cell in vivo. In some embodiments, the detectingcomprises detecting MECT1-MAML2 mRNA or detecting MECT1-MAML2 protein.

DESCRIPTION OF THE FIGURES

[0015]FIG. 1, Panel A depicts the spectral karyotyping of MEC tumor cellline showing normal chromosome 11 and the reciprocal t(11;19) with Der.11 and Der. 19. The display color (left), DAPI G-banding-like (middle)and classification representation (right) are shown for each chromosome.For the classification representation, blue represents chromosome 11 andgreen represents chromosome 19. FIG. 1, Panel B provides data from FISHanalysis showing overlapping hybridization of the immediately adjacentRP11-676L3 (green) and RP11-16K5 (red) BAC clones on the normalchromosome 11 at band q21, while mapping of RP11-16K5 to Der. 19 andRP11-676L3 to the Der. 11 chromosomes. Note the weak signal of theRP11-676L3 probe that maps with RP11-16K5 on the Der. 19 chromosome(arrow), which localizes the 11q21 chromosomal breakpoint near thetelomeric end of RP11-676L3.

[0016]FIG. 2, Panel A provides a schematic representation of the partialgenomic structure of the MAML2 and MECT1 genes and the approximatelocation of the translocation breakpoint. FIG. 1, Panel B shows theresults of an RT-PCR analysis using MECT1 exon 1 (sense) and MAML2 exon2 (antisense) oligonucleotides as indicated. Lanes 1, 8, and 9correspond to size markers, lanes 2 and 10 correspond to negativecontrol reactions, lanes 3-5 and 11-13 correspond to reactions performedwith RNA derived from MEC tumors and lanes 6 and 7 correspond toreactions performed with RNA derived from non-MEC tumors.

[0017]FIG. 3 provides a schema depicting the growing Mastermind-likegene family and a minimal hypothetical sequence motif A conservedamino-terminal, highly basic domain within MAML2 (GenBank: AY040322),MAML1 (GenBank: XM 011324), an anonymous sequence, KIAA1819 (GenBank:AB058719), and Mastermind (MAM; GenBank: X5425 1) is indicated by ablack rectangle, while an amino-terminal basic domain within the C.elegans LAG-3A gene product is depicted with a hatched rectangle. Theminimal, hypothetical consensus sequences within the NOTCH bindingregion is shown and provided as: MAML2, SEQ ID NO:5; MAML, SEQ ID NO:6,KIAA1819, SEQ ID NO:7; MAM, SEQ ID NO:8, and LAG-3A, SEQ ID NO:9.Identical and conserved amino acid residues of MAM and LAG-3A are boxed.

[0018]FIG. 4 shows the results of a fluorescence analysis ofMECT1-MAML2. In Panel A, COS7 cells were transiently transfected withplasmids expressing GFP-tagged MECT1-MAML2 or FLAG-tagged MAML2proteins. In Panel B, COS7 cells were transiently co-transfected withGFP-tagged ICN1 and either an empty pFLAG-CMV-2 vector (BG) (column 1),FLAG-tagged MECT1-MAML2 (column 2), or FLAG-tagged MAML2 (column 3). Inboth panels, staining was done with an anti-FLAG antibody, while DAPIstaining was used to identify the cell nuclei.

[0019]FIG. 5 shows the results of biochemical analyses of MECT1-MAML2.In Panel A, COS7 cells were co-transfected with different combinationsof FLAG-tagged MAML2, FLAG-tagged MECT1-MAML2, HA-tagged ICN1, andMyc-tagged CSL as indicated. Anti-FLAG immunoprecipitates (IP) or wholecell lysates (WCL) were immunoblotted (WB) with anti-FLAG, anti-HA, oranti-myc antibodies. In Panel B, U2OS cells were transfected with 0.5 μgpG5luc (containing four GAL4 binding sites and a firefly luciferasereporter), 25 ng pRL-TK plasmid encoding Renilla luciferase and 0.5 μgof GAL4 DNA binding domain (BD) only, or BD fused to MECT1-MAML2, MAML2,or MAML2 (174-1153). Activity was normalized to Renilla luciferase.

[0020]FIG. 6 indicates that MECT1-MAML2 activation is independent ofJagged2 stimulation and CSL binding sites. In Panel A, U2OS cells wereco-transfected with 0.5 μg of the HES1-luc promoter construct, 25 ngpRL-TK plasmid encoding Renilla luciferase, and increasing amounts ofpFLAG-CMV2 plasmids (in μg) encoding MAML2 (M2), MECT1-MAML2 (M-M2), andMAML2 (172-1153) (ΔM2). 20 h post-transfection, 1×10⁵ NIH 3T3 cellsexpressing Jagged2 or NIH 3T3 cells infected with empty pBABE virus wereadded to each well and luciferase activity was measured 24 h later.Panel B, shows the results of the same experimental design applied to anHES1 promoter lacking two CSL binding sites (HES1-Δ).

[0021]FIG. 7 indicates that MECT1-MAML activation shows narrow promoterspecificity. U20S cells were transfected with 0.5 μg of the indicatedpromoter/reporter constructs, 25 ng pRL-TK plasmid encoding Renillaluciferase, and increasing amounts of the indicated MECT1-MAML2plasmids.

[0022]FIG. 8 indicates that MECT1-MAML2 and MECT1-MAML1, but notMECT1-VP16, mediates CSL-independent and ICN-independent activation.Panel A shows activation of the 4×CSL-wt-luc plasmid, Panel B showsactivation of the CSL-mutant-luc (mt) plasmid, and Panel C showsactivation of the 4×CSL-wt-luc and HES1-luc luciferase reporter plasmidsas induced by varying amounts of co-transfected ICN1, M2, M-M2,MECT1-MAML1 (M-M1) or MECT1-VP16 (M-VP) plasmids as indicated.

[0023]FIG. 9 shows induction of HES1 mRNA by the MECT1-MAML2 product invivo. Panel A provides the results of an RT-PCR analysis using total RNAextracted from immortalized, normal parotid cells (HS4) or tumor cells(H292). Panel B provides the results of an RT-PCR analysis using totalRNA extracted from transiently transfected HS4 cells with either vectoralone (vec) or MECT1-MAML2 (M-M2). The 28S ribosomal signals from theRNA samples are indicated.

DESCRIPTION OF THE INVENTION

[0024] The present invention provides methods and compositions for thediagnosis and treatment of cancer, including cancers involving the NOTCHpathway. In particular, the present invention provides methods andcompositions for the diagnosis of mucoepidermoid carcinoma, the mostcommon malignant salivary gland tumor. The present invention furtherprovides methods and compositions for the diagnosis of other tumorsassociated with the t(11;19)(q14-21;12-13) translocation.

[0025] As indicated above, mucoepidermoid carcinoma (MEC) is the mostcommon malignant human salivary gland tumor which can arise from bothmajor (parotid) and minor salivary glands, including serous/mucousglands within the pulmonary tracheobronchial tree. Recently, cytogeneticstudies have demonstrated a t(11;19)(q14-21;p12-13) translocation in 12patients with MEC obtained from different tissue sites. In five of thesecases, the t(11;19) was the sole chromosomal alteration (Johansson, etal., Cancer Genet. Cytogenet., 80:85-86 [1995]; Horsman et al., CancerGenet. Cytogenet., 80:165-166 [1995]; El-Naggar et al., Cancer Genet.Cytogenet., 87:29-33 [1996]; Dahlenfors et al., Cancer Genet.Cytogenet., 79:188 [1995]; and Dahlenfors et al., Hereditas 120:287-288[1994]). In addition, the same translocation event has been detected infour patients with Warthin's tumor, a distinct histologic type ofparotid salivary gland tumor; in one case, it was the sole chromosomalabnormality (Bullerdiek et al., Cancer Genet. Cytogenet., 35:129-32[1988]; Nordkvist et al., Cancer Genet. Cytogenet., 76:129-135 [1994];and Martins et al., Oral Oncol., 33: 344-347 [1997]).

[0026] As discussed in greater detail below, by providing theidentification of a tumor-specific, mutant MECT1/MAML2 fusion protein,the present invention provides methods and compositions applicable tocell biology, genetics, and diagnosis of this important class of humansalivary/mucous gland tumors. While the classification of human salivarygland tumors has historically relied on histopathology by lightmicroscopy, the distinction between benign and malignant tumors ofdifferent subtypes is often difficult, at least partially due to thepresence of mixed epithelial cell types in these tumors (See,Calcaterra, supra) and to the common use of fine needle aspiration forclinical diagnosis. Pleomorphic adenoma (or benign mixed tumor) is themost common benign tumor arising from the parotid and other upperaerodigestive tract glands and has been recently associated withchromosomal rearrangements at 8q12 or 12q13-15 that activate the PLAG1and/or HMGIC gene families, respectively (Kas et al., Nat. Genet.,15:170-174 [1997]; and Schoenmakers et al., Nat. Genet., 10:436-444[1995]). In contrast, MEC tumors and Warthin's tumor are the most commonmalignant tumors arising from the parotid gland, as well as from minorserous/mucous glands scattered throughout the upper aerodigestive tract.

[0027] During the development of the present invention, it wasdetermined that these tumors with a t(11;19)(q14-21;p12-13) arecharacterized by the expression of a unique chimeric MECT1/MAML2 productwhich may be pathogenic for these specific tumors. Finally, adenoidcystic carcinomas are the second most common type of malignant salivarygland tumor and are associated with several different chromosomalalterations including the detection of a del(6q) andt(6;9)(q21-24;p13-23), but do not demonstrate the t(11;19) rearrangementpresent in MEC samples (Jin et al., Genes Chromosomes Cancer 30:161-17[2001]). Thus, the present invention provides means for a new frameworkfor the molecular diagnosis of human MEC and Warthin's tumors, as wellas providing means for pre- and/or post-surgical mapping of tumormargins to improve local control and to help the medical practitionerdecide whether there is a need for adjuvant therapies. In addition, thepresent invention provides a means to facilitate the determination ofwhether these mixed lineage tumors arise from ductal epithelial stemcells or from specific committed epithelial cells, and thereby providesa new approach for understanding the biological basis for these oftenlocally recurrent tumors.

[0028] During the development of the present invention, spectralkaryotyping was performed on two independent pulmonary MEC tumor celllines, namely NCI-H292 and H3118. As indicated in FIG. 1, Panel A,evidence for reciprocal t(11;19) translocation was observed in bothcases. Using multiple bacterial artificial chromosome (BAC) probeslocated at chromosome 11q14-21 for fluorescence in situ hybridization(FISH) analysis, it was determined that the immediately adjacent BACclones, RP11-676L3 and RP11-16K5, mapped together near band q21 on thenormal chromosome 11 (See, FIG. 1, Panel B). In contrast, RP11-676L3hybridized to the Der. chromosome 11, while RP11-16K5 mapped to the Der.19 chromosome in both pulmonary MEC tumor cell lines (See, FIG. 1, PanelB). In addition, a very faint, but specific, signal from BAC RP11-676L3was also detected on the Der. 19 chromosome indicating that thetranslocation breakpoint was located close to the telomeric end ofRP11-676L3. Inspection of the genomic sequence in this region withinchromosome 11q21 identified an open reading frame approximately 20 kbfrom the telomeric end of RP11-676L3, which was contained within ananonymous mRNA sequence (designated “KIAA1819”). Protein blast searchanalysis (Altschul et al., J. Mol. Biol., 215:403-410 [1990]; andAltschul et al., Nucleic Acids Res., 25:3389-3402 [1997]) demonstratedthat this gene shared similarity with Drosophila Mastermind (MAM), andwith a recently identified Mastermind-like1 (MAML1) gene on humanchromosome 5 that encodes a transcriptional co-activator for NOTCHreceptors (Artavanis-Tsakonas et al., Science 268:225-232 [1995]; Xu etal., Genes Dev., 4, 464-475 [1990]; and Wu et al., Nat. Genet.,26:484-489 [2000]). Accordingly, this related novel gene was designatedas MAML2.

[0029] Genomic blast search analyses revealed that the novel MAML2 genecontains 5 exons and spans 340 kb at human 11q21. In addition, it wasobserved that the MAML2 exon 1 was contained within the BAC RP11-16K5(which mapped to the Der 19), while exon 2 was separated by a 270 kbintron 1, confirming that MAML2 was disrupted by a chromosomalbreakpoint near the 3′ end of the large MAML2 intron 1 (See, FIG. 2,Panel A). 5′ rapid amplification of cDNA ends (RACE), using RNAextracted from both MEC samples revealed a single amplified productusing first-strand cDNA primed independently from either the polyA tailor from a specific MAML2 exon 2 sequence. Direct nucleotide sequencing,demonstrated a chimeric mRNA species representing exon 1 of a novel geneat 19p12-13 (MECT1) fused in-frame to MAML2 exons 2-5 (See, FIG. 2,Panel B).

[0030] To confirm the expression of the MECT1-MAML2 chimeric product,RT-PCR was done using gene-specific primer pairs from MECT1 exon 1 andMAML2 exon 2 with tumor RNA isolated from five different tumors: threeprimary tumor biopsy samples from patients with either bronchopulmonary,lingual, or parotid MEC (MEC A-C) and two cultured tumor cell lines(H292 and H3118). The identical 203 bp mutant chimera was detected inall five MEC samples (See, FIG. 2, Panel B), but not in 20 differentnon-MEC tumors. Additionally, by using different oligonucleotideprimers, the full-length 3.7 kb MECT1/MAML2 fusion species was alsodetected in the MEC samples. Since MEC-C gave a relatively weak signalusing the semi-quantitative RT-PCR technique, a RNase protection assaywas done, confirming the presence of steady-state levels of theMECT1-MAML2 chimera in MEC-B and MEC-C. Taken together, theseobservations indicate that the MECT1/MAML2 chimeric protein is amolecular marker for MEC tumors.

[0031] Using multiple different primer sets for MAML2 and MECT1,expression of the hypothetical, reciprocal chimeric product encodingMAML2 exon 1/MECT1 exons 2-18 was not observed. This is consistent withthe expression of normal MECT1 mRNA, but not MAML2 in the MEC celllines. An explanation for the lack of this reciprocal product includesthe possibility that the MAML2 promoter is inactive in salivary andserous/mucous gland tissues, as well as the observation that thischimeric intron 1 would span approximately 300 kb which may be beyondthe limits for proper splicing of a non-native intron sequence. However,an understanding of the mechanism(s) is not necessary in order to makeand use the present invention.

[0032] Inspection of the MECT1 gene at 19p12-13 showed that it contains18 exons and has a duplicated gene sequence at chromosome 2p16.2 whichis contemplated as being a pseudogene. The translated MECT1 proteinsequence has no previously defined functional motifs and shows aminoacid similarities within discrete domains to only two other anonymoustranscripts in the NCBI database. In contrast, the predicted sequence ofMAML2 showed 22% identity and 34% similarity over 1189 residues with theMastermind-like homolog, MAML1. Drosophila Mastermind (MAM) is one ofthe original “neurogenic” genes, and has been identified as a componentof the NOTCH signaling pathway. In particular, exon 1 of MAML2 ispredicted to encode the complete conserved ‘basic region’ found near theamino-terminus of MAM and MAML1 (See, FIG. 3). This charged domain hasbeen shown to bind to the ankyrin repeats of the intracellular NOTCHreceptor domain (ICN) (See e.g., Artavanis-Tsakonas et al., Science268:225-232 [1995]; Wu et al., Nat. Genet., 26:484-489 [2000]; Aster etal., Mol. Cell. Biol., 20:7505-7515 [2000]; and Petcherski and Kimble,Nature 405:364-8 [2000]). In addition, Psi-Blast protein alignment(Altschul et al., Nucleic Acids Res., 25:3389-402 [1997]) showed apotential, minimal NOTCH binding domain within MAML2, that is alsopresent within an amino-terminal region of the LAG-3 gene product(Petcherski and Kimble, Nature 405:364-368 [2000]). This hypotheticalalignment with Lag-3, however, lacks statistical significance andremains to be confirmed by protein binding assays.

[0033] The mammalian MAML1 has been shown to function as atranscriptional co-activator for Notch, forming a complex in the nucleuswith the intracellular domain of an activated Notch receptor (ICN) andthe bifunctional transcription factor CBF1/Su(h)/Lag1 (CSL; Wu et al.,supra [2000]). Formation of the ICN/CSL/MAML1 complex activates thetranscription of Notch target genes, including HES1, the bestcharacterized member of the HES family (mammalian homologues ofDrosophila Hairy and Enhancer of Split genes; Artavanis-Tsakonas et al.,supra [1995]; and Kojika and Griffin, Exp. Hematol., 29:1041-1052[2001]).

[0034] Interestingly, the ectopic expression of an in vitro MAML1 mutantgene, lacking the amino-terminal NOTCH binding domain, but retaining thecarboxy-terminal transactivation domain (TAD), was recently shown toexhibit a dominant-negative phenotype by inhibiting the ability of ICNto activate its normal downstream target, the HES1 promoter (Wu et al.,supra [2000]). During the development of the present invention, it wasdemonstrated that the t(11;19)(q14-21;p12-13) alteration found in themost common type of malignant salivary gland tumors results in the invivo expression of a fusion product that would also selectively lack theamino-terminal NOTCH binding domain, but retain the TAD andglutamine-rich domains that are conserved in the Mastermind-like andLag-3 gene family.

[0035] To test the function of MECT1-MAML2 and MAML2 in Notch signaling,the sub-cellular localization of these proteins was compared. Using agreen fluorescent protein (GFP) tag or fluorescent anti-FLAG, bothproteins were observed to co-localize in a nuclear structure with aspeckled staining pattern (See, FIG. 4, Panel A), identical to thatpreviously described for MAML1 (Wu et al., supra [2000]). As shown inFIG. 4, Panel B, transfected ICN1-GFP localized to the nucleus in adiffuse pattern. However, co-expression of either MECT1-MAML2 or MAML2,was able to induce re-localization of ICN1 from a diffuse nuclearpattern into a distinct, speckled nuclear structure (See, FIG. 4, PanelB). In addition, both MECT1-MAML2 and MAML2 co-localized with ICN1 inthese nuclear bodies (See, FIG. 4, Panel B ‘merge’). Immunoprecipitationwas performed to determine whether MECT/MAML2 physically interacted withICN1. As shown in FIG. 5, Panel A, both MAML2 and MECT1-MAML2co-immunoprecipitated with ICN1, although the MECT1-MAML2 interactionwith ICN appeared to be weaker. However, only MAML2 and not MECT1-MAML2,co-immunoprecipitated in a multiprotein complex with CSL and ICN1.

[0036] A transcriptional activation domain (TAD) was previously mappedto the carboxy-terminal region of MAML1 (Wu et al., supra [2000]). Todetermine whether MAML2 and MECT1-MAML2 also contain a TAD, theappropriate cDNAs were fused with the Gal4 DNA binding domain (BD). Asshown in FIG. 5, Panel B, both MAML2 and MECT1-MAML2 encode a functionalTAD. In addition, the carboxy-terminal component of the MAML2 (exons2-5; aa172-1153) also was observed to retain a high level of TADactivity (See, FIG. 5, Panel B).

[0037] The ability of MECT1-MAML2 and wild-type MAML2 to participate inNotch signaling was further evaluated by examining activation of theNotch target gene, HES1. As shown in FIG. 6, Panels A and B, MAML2enhanced the Notch ligand (Jagged2)-induced activation of the HES1promoter, but did not enhance activation of a HES1 promoter lacking thetwo endogenous CSL binding sites (HES1-Δ). Surprisingly, activation ofthe HES1 promoter by the MECT1-MAML2 chimera was independent of Notchligand stimulation and was independent of the CSL binding sites withinthe HES1 promoter (HES1-Δ). The truncated MAML2 (aa172-1153), whichretained the TAD but which lacks the N-terminal exon 1 sequences (ΔM2),failed to activate HES1. MECT1-MAML2 showed mild activation of the HES7promoters in U20S cells, HeLa cells and 293 cells, but did not activatetranscription of promoters from the telomerase (hTERT), cyclin dependentkinase inhibitors p21 or p27, or the HES5 genes (See, FIG. 7). Thesefindings are indicative of a narrow promoter specificity for theMECT1-MAML2 product. Moreover, the observation that MECT1-MAML2 isunable to form a complex with CSL and can activate the HES1 promoterindependent of CSL, indicates that MECT1-MAML2 must function throughanother unknown binding site on the HES1 promoter. To confirm thatMECT1-MAML2 acts independently of CSL, the transcriptional activation ofan artificial promoter containing 4 copies of either a wild type or amutant CSL binding site in front of an SV40 promoter (4×CSL-wt-luc and4×CSL-mt-luc, respectively) was tested. Previously, transfection of ICNhad been shown to activate the wild type promoter in a CSL dependentmanner (Hsieh et al., Mol. Cell Biol., 16:952-959 [1996]). As shown inFIG. 8 Panel A, MAML2 (M2) amplified the ICN1-induced activation of thewild type CSL promoter, while MECT1-MAML2 had no stimulatory effect orwas inhibitory. No activation was observed with the mutant CSL promoter(See, FIG. 8, Panel B). To examine the contribution of the MAML-likeTAD, the MAML2 sequence was replaced with either the equivalent sequencefrom MAML1 (MECT1-MAML1; M-M1) or with an unrelated transcriptionalactivator, VP16 (MECT1-VP16; M-VP). As shown in FIG. 8, Panel C,MECT1-MAML1, like MECT1-MAML2, could activate the HES1 promoterindependently of ICN1, while MECT1-VP16 had a negligible effect on theHES1 promoter.

[0038] Confirming the effect of MECT1-MAML2 on the HES1 promoter invitro, a high HES1 mRNA expression level was detected in the pulmonaryMEC line (H292). As shown in FIG. 9, Panel A, this contrasted with thelow or absent HES1 mRNA expression level observed in normal,immortalized parotid gland cells (HS4). Transient transfection of theMECT1-MAML2 cDNA into the normal HS4 cells, however, resulted in a rapidinduction of HES1 mRNA at 48 hrs as compared to mock-transfected HS4cells (See, FIG. 9, Panel B).

[0039] While chromosomal rearrangements are commonly observed inhematopoietic and mesenchymal stromal tumors, <1% of all epithelialcarcinomas show a recurrent, pathogenic chromosomal alteration(Mitelman, Mutat. res., 462:247-253 [2000]). MEC, therefore, representsa new epithelial tumor model system, in which a chimeric gene productdisrupts Notch signaling via a novel CSL-independent mechanism. In thecase of t(7;9) and T-ALL, the proposed consequence of the translocationis to both deregulate the expression of the mutant Notch1 receptor genewith T-cell receptor βpromoter/enhancer sequences and to express atruncated Notch molecule that can localize to the nucleus andconstitutively activate HES family members independent of Notch ligand(Aster and Pear, Curr. Opin. Hematol., 8:237-244 [2001]; and Allman etal., Cell 109S:S1-11 [2002]). While Notch receptors can regulate thedifferentiation and development of diverse cell lineages, the mechanismsunderlying T-cell leukemogenesis are still unknown. During developmentof the present invention, it has now been demonstrated that the t(11;19)alteration linked with MEC can result in the deregulated expression of amutant Notch co-activator which in turn can constitutively activate HES1gene expression. Thus, disruption of NOTCH signaling and/or otherfunctions of the novel MECT1/MAML2 product are contemplated to be anessential component in the genesis of epithelial pulmonary MEC tumors,as well as other human salivary gland tumors associated with thet(11;19) alteration. Therefore, the present invention provides methodsand compositions for the diagnosis and treatment for such tumors.

[0040] Definitions

[0041] As used herein, the terms “purified” and “to purify” refer to theremoval of one or more (undesired) components from a sample. Forexample, where recombinant polypeptides are expressed in bacterial hostcells, the polypeptides are purified by the removal of host cellproteins thereby increasing the percent of recombinant polypeptides inthe sample.

[0042] As used herein, the term “partially purified” refers to theremoval of a moderate portion of the contaminants of a sample to theextent that the substance of interest is recognizable by techniquesknown to those skilled in the art as accounting for a measurable amountof the mixture.

[0043] As used herein, the term “substantially purified” refers tomolecules, (e.g., nucleic or amino acid sequences) that are removed fromtheir natural environment, isolated or separated, and are at least 60%free, preferably 75% free, and more preferably 90% free from othercomponents with which they are naturally associated. Furthermore, an“isolated polynucleotide” encompasses a substantially purifiedpolynucleotide.

[0044] As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, ovines, bovines, ruminants, lagomorphs, porcines, caprines,equines, canines, felines, aves, etcetera. In some embodiments, the term“subject” refers to the animal from which a biopsy is obtained fortesting. Typically, the terms “subject” and “patient” are usedinterchangeably in reference to a human subject.

[0045] As used herein, the term “subject suspected of having cancer”refers to a subject that presents one or more symptoms indicative of acancer (e.g., a noticeable lump or mass) or is being screened for acancer (e.g., during a routine physical). A subject suspected of havingcancer may also have one or more risk factors. A subject suspected ofhaving cancer has generally not been tested for cancer. However, a“subject suspected of having cancer” encompasses an individual who hasreceived an initial diagnosis (e.g., a CT scan showing a mass) but forwhom the stage or type of cancer is not known. The term further includespeople who once had cancer (e.g., an individual in remission).

[0046] As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental expose, previous incidents of cancer,pre-existing non-cancer diseases, and lifestyle.

[0047] As used herein, the term “characterizing cancer in subject”refers to the identification of one or more properties of a cancer in asubject, including but not limited to, the presence of benign,pre-cancerous or cancerous tissue, the stage of the cancer, and thesubject's prognosis. Cancers may be characterized by the identificationof the expression of one or more cancer marker genes, including but notlimited to, the cancer markers disclosed herein.

[0048] As used herein, the term “cancer marker genes” refers to a genewhose expression level, alone or in combination with other genes, iscorrelated with cancer or prognosis of cancer. The correlation mayrelate to either an increased or decreased expression of the gene. Forexample, the expression of the gene may be indicative of cancer, or lackof expression of the gene may be correlated with poor prognosis in acancer patient. In preferred embodiments, cancer marker expressionrefers to MECT1-MAML2 expression, which may be characterized using anysuitable method, including but not limited to, those described inillustrative Examples 2-4 below.

[0049] As used herein, the term “a reagent capable of specificallydetecting” refers to reagents used to monitor the presence and/orquantity of a gene or gene product of interest (e.g., including but notlimited to the MECT1-MAML2 chimera of the present invention). Examplesof suitable reagents include but are not limited to, nucleic acid probescapable of specifically hybridizing to the gene of interest, PCR primerscapable of specifically amplifying the gene of interest, and antibodiescapable of specifically binding to proteins expressed by the gene ofinterest

[0050] As used herein, the terms “detecting a change in gene expressionrelative to” and “detecting a decrease or an increase in gene expressionrelative to” refer to measuring the level of expression of a gene (e.g.,the level of mRNA or protein) relative to the level in a control sample(e.g., sample lacking a test compound). Gene expression can be measuredusing any suitable method, including but not limited to, those describedherein.

[0051] The term “screening” refers to the examination of a sample for agenotype or phenotype of interest. In preferred embodiments, thegenotype of interest comprises the t(11;19)(q14-21;p12-13)translocation, while the phenotype comprises the expression of theMECT1-MAML2 chimera.

[0052] The term “tissue sample” refers to specimen comprises cells. Inpreferred embodiments, the specimen comprises “biopsy material.” Theterm “biopsy” refers to specimen (e.g., salivary gland tissue) collectedfrom a subject for further analysis to establish a diagnosis (e.g.,mucoepidermoid carcinoma). Biopsies can be accomplished with a biopsyneedle (passed through the skin into the organ in question) or by opensurgical incision.

[0053] As used herein, the term “salivary gland tumor” refers to anabnormal mass of tissue of any of the saliva-secreting exocrine glandsof the oral cavity, that results from excessive cell division (e.g.,neoplasm). Salivary gland tumors include but are not limited tomucoepidermoid cancer, pleomorphic adenoma and adenoid cystic carcinoma.The term “mucoepidermoid cancer” refers to a malignant epithelial tumourof glandular tissue, especially the salivary glands, characterised byacini with mucus-producing cells and by the presence of malignantsquamous elements. The term “pleomorphic adenoma” refers to a mixedtumour of the salivary gland composed of salivary gland epithelium andfibrous tissue with mucoid or cartilaginous areas. The terms “adenoidcystic carcinoma” and “cylindromatous carcinoma” refer to carcinomacharacterised by large epithelial masses containing round, glandlikespaces or cysts which frequently contain mucus or collagen and arebordered by a few or many layers of epithelial cells without interveningstroma, thereby forming a cribriform pattern.

[0054] As used herein, the term “instructions for using said kit forscreening a tissue sample” refers to the directions for using thereagents contained in the kit for the detection of at(11;19)(q14-21:p12-13) translocation and/or a MECT1-MAML2 chimera. Insome embodiments, the instructions further comprise the statement ofintended use required by the U.S. Food and Drug Administration (FDA) inlabeling in vitro diagnostic products. The FDA classifies in vitrodiagnostics as medical devices and requires that they be approvedthrough the 510(k) procedure. Information required in an applicationunder 510(k) includes: 1) The in vitro diagnostic product name,including the trade or proprietary name, the common or usual name, andthe classification name of the device; 2) The intended use of theproduct; 3) The establishment registration number, if applicable, of theowner or operator submitting the 510(k) submission; the class in whichthe in vitro diagnostic product was placed under section 513 of the FD&CAct, if known, its appropriate panel, or, if the owner or operatordetermines that the device has not been classified under such section, astatement of that determination and the basis for the determination thatthe in vitro diagnostic product is not so classified; 4) Proposedlabels, labeling and advertisements sufficient to describe the in vitrodiagnostic product, its intended use, and directions for use. Whereapplicable, photographs or engineering drawings should be supplied; 5) Astatement indicating that the device is similar to and/or different fromother in vitro diagnostic products of comparable type in commercialdistribution in the U.S., accompanied by data to support the statement;6) A 510(k) summary of the safety and effectiveness data upon which thesubstantial equivalence determination is based; or a statement that the510(k) safety and effectiveness information supporting the FDA findingof substantial equivalence will be made available to any person within30 days of a written request; 7) A statement that the submitterbelieves, to the best of their knowledge, that all data and informationsubmitted in the premarket notification are truthful and accurate andthat no material fact has been omitted; 8) Any additional informationregarding the in vitro diagnostic product requested that is necessaryfor the FDA to make a substantial equivalency determination. Additionalinformation is available at the Internet web page of the U.S. FDA.

[0055] The term “translocation” refers to a rearrangement of achromosome in which a segment is moved from one location to another,either within the same chromosome or to another chromosome. The issometimes reciprocal, when one fragment is exchanged for another.

[0056] As used herein, the terms “chimera” and “chimeric” refer to amolecule (e.g., gene, transcript or protein) composed of parts that areof different origin and are seemingly incompatible. In preferredembodiments of the present invention the term “chimera” is used inreference to the MECT1-MAML2 chimera formed as a results of at(11;19)(q14-21:p12-13) translocation. The predicted amino acid sequenceof the MECT1-MAML2 chimera is set forth as SEQ ID NO;12.

[0057] As used herein, the term “gene expression” refers to the processof converting genetic information encoded in a gene into RNA (e.g.,mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e.,via the enzymatic action of an RNA polymerase), and for protein encodinggenes, into protein through “translation” of mRNA. Gene expression canbe regulated at many stages in the process. “Up-regulation” or“activation” refers to regulation that increases the production of geneexpression products (i.e., RNA or protein), while “down-regulation” or“repression” refers to regulation that decrease production. Molecules(e.g., transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

[0058] As used herein, the term “antibody” refers a glycoproteinproduced by B cells and plasma cells that binds with high specificity toan antigen (usually, but not always, a peptide) or a structurallysimilar antigen, that generated its production. Antibodies may beproduced by any of the known methodologies and may be either polyclonalor monoclonal, and may be of any class (e.g., IgG, IgM, IgA, IgE, IgD).

[0059] “Wild-type,” as used herein, refers to a gene or gene productwhich has the characteristics of that gene or gene product when isolatedfrom a naturally occurring source. A wild-type gene is that which ismost frequently observed in a population and is thus arbitrarilydesigned the “normal” or “wild-type” form of the gene.

[0060] “Mutant,” as used herein, refers to any changes made to awild-type nucleotide sequence, either naturally or artificially, thatproduces a translation product that functions with enhanced or decreasedefficiency in at least one of a number of ways including, but notlimited to, specificity for various interactive molecules, rate ofreaction and longevity of the mutant molecule.

[0061] “Staining,” as used herein, refers to any number of processesknown to those in the field that are used to better visualize a specificcomponent(s) and/or feature(s) of a cell or cells.

[0062] The terms “cancerous” and “cancer cell” refer to a cellundergoing early, intermediate or advanced stages of multi-stepneoplastic progression as known in the art (See e.g., Pitot, inFundamentals of Oncology, Marcel Dekker (Ed.), New York, pp. 15-28[1978]). The microscopic features of early, intermediate and advancedstages of neoplastic progression have been described. Cancer cells ateach of the three stages of neoplastic progression generally haveabnormal karyotypes, including translocations, inversion, deletions,isochromosomes, monosomies, and extra chromosomes. A cell in the earlystages of malignant progression is referred to as “hyperplastic cell”and is characterized by dividing without control and/or at a greaterrate than a normal cell of the same cell type in the same tissue.Proliferation may be slow or rapid but continues unabated. A cell in theintermediate stages of neoplastic progression is referred to as a“dysplastic cell.” A dysplastic cell resembles an immature epithelialcell, is generally spatially disorganized within the tissue and has lostits specialized structures and functions. For example, during theintermediate stages of neoplastic progression, an increasing percentageof the epithelium becomes composed of dysplastic cells. “Hyperplastic”and “dysplastic” cells are referred to as “pre-neoplastic” cells. In theadvanced stages of neoplastic progression a dysplastic cell become a“neoplastic” cell. Neoplastic cells are typically invasive. Thus, theyeither invade adjacent tissues, or are shed from the primary site andcirculate through the blood and lymph to other locations in the bodywhere they initiate secondary cancers. The term “cancer” or “neoplasia”refers to a plurality of cancer cells.

[0063] “Nucleic acid sequence,” “nucleotide sequence” and“polynucleotide sequence” as used herein, refer to an oligonucleotide orpolynucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin which may be single- or double-stranded, andrepresent the sense or antisense strand.

[0064] As used herein, the terms “oligonucleotides” and “oligomers”refer to a nucleic acid sequence of at least about 10 nucleotides and asmany as about 60 nucleotides, preferably about 15 to 30 nucleotides, andmore preferably about 20-25 nucleotides, which can be used as a probe oramplimer.

[0065] A “variant” of a nucleotide sequence is defined as a nucleotidesequence which differs from the referenced, parent or wild typenucleotide sequence (e.g., by having one or more deletions, insertions,or substitutions that may be detected using hybridization assays orusing DNA sequencing). Included within this definition is the detectionof alterations to the genomic sequence of the nucleotide sequence. Forexample, hybridization assays may be used to detect alterations in: (1)the pattern of restriction enzyme fragments capable of hybridizing to agenomic sequence of the first nucleotide sequence (i.e., RFLP analysis);(2) the inability of a selected portion of the first nucleotide sequenceto hybridize to a sample of genomic DNA which contains the firstnucleotide sequence (e.g., using allele-specific oligonucleotideprobes); and (3) improper or unexpected hybridization, such ashybridization to a locus other than the normal chromosomal locus for thefirst nucleotide sequence (e.g., using fluorescent in situ hybridization(FISH) to metaphase chromosomes spreads, etc.). One example of a variantis a mutated wild type sequence.

[0066] The term “portion” when used in reference to a nucleotidesequence refers to fragments of that nucleotide sequence. The fragmentsmay range in size from 5 nucleotide residues to the entire nucleotidesequence minus one nucleic acid residue.

[0067] DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotide is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring. An end of an oligonucleotide is referred toas the “3′ end” if its 3′ oxygen is not linked to a 5′ phosphate ofanother mononucleotide pentose ring. As used herein, a nucleic acidsequence, even if internal to a larger oligonucleotide, also may be saidto have 5′ and 3′ ends. In either a linear or circular DNA molecule,discrete elements are referred to as being “upstream” or 5′ of the“downstream” or 3′ elements. This terminology reflects thattranscription proceeds in a 5′ to 3′ direction along the DNA strand. Thepromoter and enhancer elements which direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

[0068] The term “recombinant DNA molecule” as used herein refers to aDNA molecule which is comprised of segments of DNA joined together bymeans of molecular biological techniques.

[0069] The term “recombinant protein” or “recombinant polypeptide” asused herein refers to a protein molecule which is expressed using arecombinant DNA molecule. As used herein, the terms “vector” and“vehicle” are used interchangeably in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another.

[0070] As used herein, the term “vector” is used in reference to nucleicacid molecules that transfer DNA segment(s) from one cell to another.The term “vehicle” is sometimes used interchangeably with “vector.”Vectors are often derived from plasmids, bacteriophages, or plant oranimal viruses.

[0071] The terms “expression vector,” “expression construct,”“expression cassette” and “plasmid,” as used herein refer to arecombinant nucleic acid molecule containing a desired coding sequenceand appropriate nucleic acid sequences necessary for the expression ofthe operably linked coding sequence in a particular host organism. Thesequences may be either double or single-stranded. Nucleic acidsequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

[0072] The terms “in operable combination,” “in operable order,” and“operably linked” as used herein refer to the linkage of nucleic acidsequences in such a manner that a nucleic acid molecule capable ofdirecting the transcription of a given gene and/or the synthesis of adesired protein molecule is produced. The terms also refer to thelinkage of amino acid sequences in such a manner so that a functionalprotein is produced.

[0073] “Reporter construct,” “reporter gene,” and “reporter protein,” asused herein, refer to nucleic acid or amino acid sequences, asappropriate, that, when expressed in a host cell or organism, may bedetected, measured and/or quantitated.

[0074] The term “transfection” as used herein refers to the introductionof foreign nucleic acid (e.g., DNA) into cells. Transfection may beaccomplished by a variety of means known to the art including calciumphosphate-DNA co-precipitation, DEAE-dextran-mediated transfection,polybrene-mediated transfection, electroporation, microinjection,liposome fusion, lipofection, protoplast fusion, retroviral infection,biolistics (i.e., particle bombardment), and the like.

[0075] The term “stable transfection” or “stably transfected” refers tothe introduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

[0076] The term “transient transfection” or “transiently transfected”refers to the introduction of foreign DNA into a cell where the foreignDNA fails to integrate into the genome of the transfected cell. Theforeign DNA persists in the nucleus of the transfected cell for severaldays. During this time the foreign DNA is subject to the regulatorycontrols that govern the expression of endogenous genes in thechromosomes. The term “transient transfectant” refers to cells that havetaken up foreign DNA but have failed to integrate this DNA.

[0077] As used herein, the terms “complementary” or “complementarity”are used in reference to “polynucleotides” and “oligonucleotides” (whichare interchangeable terms that refer to a sequence of nucleotides)related by the base-pairing rules. For example, the sequence“5′-CAGT-3′,” is complementary to the sequence “5′-ACTG-3′.”Complementarity can be “partial” or “total.” “Partial” complementarityis where one or more nucleic acid bases is not matched according to thebase pairing rules. “Total” or “complete” complementarity betweennucleic acids is where each and every nucleic acid base is matched withanother base under the base pairing rules. The degree of complementaritybetween nucleic acid strands may have significant effects on theefficiency and strength of hybridization between nucleic acid strands.This may be of particular importance in amplification reactions, as wellas detection methods which depend upon binding between nucleic acids.

[0078] The terms “homology” and “homologous” as used herein in referenceto nucleotide sequences refer to a degree of complementarity with othernucleotide sequences. There may be partial homology or complete homology(i.e., identity). A nucleotide sequence which is partially complementary(i.e., “substantially homologous”) to a nucleic acid sequence is onethat at least partially inhibits a completely complementary sequencefrom hybridizing to a target nucleic acid sequence. The inhibition ofhybridization of the completely complementary sequence to the targetsequence may be examined using a hybridization assay (Southern orNorthern blot, solution hybridization and the like) under conditions oflow stringency. A substantially homologous sequence or probe willcompete for and inhibit the binding (i.e., the hybridization) of acompletely homologous sequence to a target sequence under conditions oflow stringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding the probe will not hybridize tothe second non-complementary target.

[0079] In preferred embodiments, an oligonucleotide sequence which is a“homolog” of a first nucleotide sequence is an oligonucleotide sequencewhich exhibits greater than or equal to 50% identity, and morepreferably greater than or equal to 70% identity, to the firstnucleotide sequence, when sequences having a length of 10 bp or largerare compared.

[0080] As used herein, the term “hybridization” is used in reference tothe pairing of complementary nucleic acids using any process by which astrand of nucleic acid joins with a complementary strand through basepairing to form a hybridization complex. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

[0081] The terms “FISH” and “fluorescence in situ hybridization” referto a physical mapping approach that uses fluorescent tags to detecthybridization of probes with metaphase chromosomes and with theless-condensed somatic interphase chromatin.

[0082] As used herein the term “hybridization complex” refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized to a solid support (e.g., anylon membrane or a nitrocellulose filter as employed in Southern andNorthern blotting, dot blotting or a glass slide as employed in in situhybridization, including FISH (fluorescent in situ hybridization)).

[0083] As used herein, the term “T_(m)” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. The equation for calculating theT_(m) of nucleic acids is well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation: T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (See e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization[1985]). Other references include more sophisticated computations thattake structural as well as sequence characteristics into account for thecalculation of T_(m).

[0084] As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Under “low stringency conditions” anucleic acid sequence of interest will hybridize to its exactcomplement, sequences with single base mismatches, closely relatedsequences (e.g., sequences with 90% or greater homology), and sequenceshaving only partial homology (e.g., sequences with 50-90% homology).Under ‘medium stringency conditions,” a nucleic acid sequence ofinterest will hybridize only to its exact complement, sequences withsingle base mismatches, and closely relation sequences (e.g., 90% orgreater homology). Under “high stringency conditions,” a nucleic acidsequence of interest will hybridize only to its exact complement, and(depending on conditions such a temperature) sequences with single basemismatches. In other words, under conditions of high stringency thetemperature can be raised so as to exclude hybridization to sequenceswith single base mismatches.

[0085] “High stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5× Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

[0086] “Medium stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5× Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 1.0×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

[0087] “Low stringency conditions” comprise conditions equivalent tobinding or hybridization at 42° C. in a solution consisting of 5×SSPE(43.8 g/l NaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to7.4 with NaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's containsper 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V;Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing ina solution comprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about500 nucleotides in length is employed.

[0088] The art knows well that numerous equivalent conditions may beemployed to comprise low stringency conditions; factors such as thelength and nature (DNA, RNA, base composition) of the probe and natureof the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, the art knows conditions that promote hybridization underconditions of high stringency (e.g., increasing the temperature of thehybridization and/or wash steps, the use of formamide in thehybridization solution, etc.) (see definition above for “stringency”).

[0089] As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target.”In contrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

[0090] As used herein, the term “primer” refers to an oligonucleotide,whether occurring naturally as in a purified restriction digest orproduced synthetically, that is capable of acting as a point ofinitiation of synthesis when placed under conditions in which synthesisof a primer extension product that is complementary to a nucleic acidstrand is induced, (i.e., in the presence of nucleotides and an inducingagent such as DNA polymerase and at a suitable temperature and pH). Theprimer is preferably single stranded for maximum efficiency inamplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

[0091] As used herein, the term “probe” refers to an oligonucleotide(i.e., a sequence of nucleotides), whether occurring naturally as in apurified restriction digest or produced synthetically, recombinantly orby PCR amplification, that is capable of hybridizing to at least aportion of another oligonucleotide of interest. A probe may besingle-stranded or double-stranded. Probes are useful in the detection,identification and isolation of particular gene sequences. It iscontemplated that any probe used in the present invention will belabeled with any “reporter molecule,” so that is detectable in anydetection system, including, but not limited to enzyme (e.g., ELISA, aswell as enzyme-based histochemical assays), fluorescent, radioactive,and luminescent systems. It is not intended that the present inventionbe limited to any particular detection system or label.

[0092] When used in reference to a double-stranded nucleic acid sequencesuch as a cDNA or genomic clone, the term “substantially homologous”refers to any probe which can hybridize either partially or completelyto either or both strands of the double-stranded nucleic acid sequenceunder conditions of low stringency as described above.

[0093] When used in reference to a single-stranded nucleic acidsequence, the term “substantially homologous” refers to any probe whichcan hybridize (i.e., it is the complement of) the single-strandednucleic acid sequence under conditions of low stringency as describedabove.

[0094] The term “heterologous nucleic acid sequence” or “heterologousDNA” are used interchangeably to refer to a nucleotide sequence which isligated to a nucleic acid sequence to which it is not ligated in nature,or to which it is ligated at a different location in nature.Heterologous DNA is not endogenous to the cell into which it isintroduced, but has been obtained from another cell. Generally, althoughnot necessarily, such heterologous DNA encodes RNA and proteins that arenot normally produced by the cell into which it is placed. Examples ofheterologous DNA include reporter genes, transcriptional andtranslational regulatory sequences, selectable marker proteins (e.g.,proteins which confer drug resistance), etc.

[0095] “Amplification” is defined herein as the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction technologies well known in the art (See e.g.,Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, Cold SpringHarbor Press, Plainview N.Y. [1995]). As used herein, the term“polymerase chain reaction” (“PCR”) refers to the methods of U.S. Pat.Nos. 4,683,195, 4,683,202, and 4,965,188, all of which are herebyincorporated by reference, and which describe a method for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of two oligonucleotide primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.”

[0096] With PCR, it is possible to amplify a single copy of a specifictarget sequence in genomic DNA to a level detectable by severaldifferent methodologies (e.g., hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular, theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications.

[0097] The terms “reverse transcription polymerase chain reaction” and“RT-PCR” refer to a method for reverse transcription of an RNA sequenceto generate a mixture of cDNA sequences, followed by increasing theconcentration of a desired segment of the transcribed cDNA sequences inthe mixture without cloning or purification. Typically, RNA is reversetranscribed using a single primer (e.g., an oligo-dT primer) prior toPCR amplification of the desired segment of the transcribed DNA usingtwo primers.

[0098] As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

[0099] As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

[0100] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to bacterial enzymes, each of which cutdouble-stranded DNA at or near a specific nucleotide sequence.

[0101] The term “Southern blot,” refers to the analysis of DNA onagarose or acrylamide gels to fractionate the DNA according to sizefollowed by transfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58 [1989]).

[0102] The term “Northern blot,” as used herein refers to the analysisof RNA by electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52[1989]).

[0103] The terms “Western blot” and “immunoblot” refer to the analysisof protein(s) (or polypeptides) immobilized onto a support such asnitrocellulose or a membrane. The proteins are run on acrylamide gels toseparate the proteins, followed by transfer of the protein from the gelto a solid support, such as nitrocellulose or a nylon membrane. Theimmobilized proteins are then exposed to antibodies with reactivityagainst an antigen of interest. The binding of the antibodies may bedetected by various methods, including the use of enzyme or radiolabeledantibodies.

[0104] As used herein, the term “microarray” refers to analysis ofindividual recombinant clones (e.g., cosmid, YAC, BAC, plasmid or othervectors) that are placed on a two-dimensional solid support (e.g.,microscope slide). Each primary clone can be identified on the supportby virtue of its location (row and column) on the solid support. Arrayedlibraries of clones can be screened with RNA obtained from a specimen ofinterest upon conjugation of a fluorochrome.

[0105] The terms “IFA” and “immunofluorescence analysis” refer to a testor technique in which one or other component of an immunologicalreaction is made fluorescent by coupling with a fluorochrome such asfluorescein, phycoerythrin or rhodamine so that the occurrence of thereaction can be detected as a fluorescing antigen-antibody complex.

[0106] As used herein, the term “probe” refers to an oligonucleotide(i.e., a sequence of nucleotides), whether occurring naturally as in apurified restriction digest or produced synthetically, recombinantly orby PCR amplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that it is detectable in any detection system, including,but not limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

[0107] As used herein, the term “sense oligonucleotide” refers to anoligonucleotide having a nucleic acid sequence which corresponds to thatof an mRNA. In contrast, the term “antisense oligonucleotide” refers toan oligonucleotide having a nucleic acid sequence which corresponds tothe complement of mRNA. the strand of DNA which is used duringtranscription to make mRNA. The mRNA made thus has the sequence of theantisense strand of DNA, and it codes for a sense strand of polypeptide(which eventually becomes a protein or part of a protein) duringtranslation.

[0108] The terms “restriction endonucleases” and “restriction enzymes,”as used herein, refer to bacterial enzymes, each of which cut double- orsingle-stranded nucleic acid at or near a specific nucleotide sequence.

[0109] As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a nucleic acid sequence comprising thecoding region of a gene (i.e. the nucleic acid sequence which encodes agene product). The coding region may be present in either a cDNA,genomic DNA or RNA form. When present in a DNA form, the oligonucleotidemay be single-stranded (i.e., the sense strand) or double-stranded.Suitable control elements (e.g., enhancers, promoters, splice junctions,polyadenylation signals, etc.) may be placed in close proximity to thecoding region of the gene if needed to permit proper initiation oftranscription and/or correct processing of the primary RNA transcript.Alternatively, the coding region utilized in the expression vectors ofthe present invention may contain endogenous enhancers, splicejunctions, intervening sequences, polyadenylation signals, or othersequences, or a combination of both endogenous and exogenous controlelements.

[0110] Transcriptional control signals in eukaryotes comprise “enhancer”elements. Enhancers consist of short arrays of DNA sequences thatinteract specifically with cellular proteins involved in transcription(See, Maniatis et al., Science 236:1237 [1987]). Enhancer elements havebeen isolated from a variety of eukaryotic sources including genes inplant, yeast, insect and mammalian cells and viruses. The selection of aparticular enhancer depends on what cell type is to be used to expressthe protein of interest.

[0111] The presence of “splicing signals” on an expression vector oftenresults in higher levels of expression of the recombinant transcript.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site (See e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, New York, pp. 16.7-16.8 [1989]). Acommonly used splice donor and acceptor site is the splice junction fromthe 16S RNA of SV40.

[0112] Efficient expression of recombinant DNA sequences in eukaryoticcells requires expression of signals directing the efficient terminationand polyadenylation of the resulting transcript. Transcriptiontermination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly A site” or “poly A sequence” as used herein denotes a DNAsequence which directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable as transcripts lacking a poly A tail areunstable and are rapidly degraded. The poly A signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly A signal is one that is found naturally at the 3′ end of the codingregion of a given gene in the genome. A heterologous poly A signal isone which is isolated from one gene and placed 3′ of another gene.

[0113] The terms “promoter,” “promoter element,” and “promoter sequence”as used herein, refer to a DNA sequence which when placed at the 5′ endof (i.e., precedes) an oligonucleotide sequence, is capable ofcontrolling the transcription of the oligonucleotide sequence into mRNA.A promoter is typically located 5′ (i.e., upstream) of anoligonucleotide sequence whose transcription into mRNA it controls, andprovides a site for specific binding by RNA polymerase and forinitiation of transcription.

[0114] The term “promoter activity” when made in reference to a nucleicacid sequence refers to the ability of the nucleic acid sequence toinitiate transcription of an oligonucleotide sequence into mRNA.

[0115] As used herein, the terms “nucleic acid molecule encoding,”“nucleotide encoding,” “DNA sequence encoding,” and “DNA encoding” referto the order or sequence of deoxyribonucleotides along a strand ofdeoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide (protein)chain. The DNA sequence thus codes for the amino acid sequence.

[0116] The term “isolated” when used in relation to a nucleic acid(e.g., “an isolated oligonucleotide”) refers to a nucleic acid sequencethat is separated from at least one contaminant nucleic acid with whichit is ordinarily associated in its natural source. Isolated nucleic acidis nucleic acid present in a form or setting that is different from thatin which it is found in nature. In contrast, non-isolated nucleic acidsare nucleic acids such as DNA and RNA which are found in the state theyexist in nature. For example, a given DNA sequence (e.g., a gene) isfound on the host cell chromosome in proximity to neighboring genes; RNAsequences, such as a specific mRNA sequence encoding a specific protein,are found in the cell as a mixture with numerous other mRNAs whichencode a multitude of proteins. However, isolated nucleic acid encodinga polypeptide of interest includes, by way of example, such nucleic acidin cells ordinarily expressing the polypeptide of interest where thenucleic acid is in a chromosomal or extrachromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid or oligonucleotide may be present in single-stranded ordouble-stranded form. Typically, isolated nucleic acid can be readilyidentified (if desired) by a variety of techniques (e.g., hybridization,dot blotting, etc.). When an isolated nucleic acid or oligonucleotide isto be utilized to express a protein, the oligonucleotide contains at aminimum, the sense or coding strand (i.e., the oligonucleotide may besingle-stranded). Alternatively, it may contain both the sense andanti-sense strands (i.e., the oligonucleotide may be double-stranded).

[0117] As used herein the term “coding region” when used in reference toa structural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

[0118] As used herein, the terms “structural gene” and “structuralnucleotide sequence” refer to a DNA sequence coding for RNA or a proteinwhich does not control the expression of other genes. In contrast, a“regulatory gene” or “regulatory sequence” is a structural gene whichencodes products (e.g., transcription factors) which control theexpression of other genes.

[0119] As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements include splicing signals,polyadenylation signals, termination signals, etc.

[0120] As used herein, the term “gene” means the deoxyribonucleotidesequences comprising the coding region of a structural gene. A “gene”may also include non-translated sequences located adjacent to the codingregion on both the 5′ and 3′ ends such that the gene corresponds to thelength of the full-length mRNA. The sequences which are located 5′ ofthe coding region and which are present on the mRNA are referred to as5′ non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences.” Intronsare segments of a gene which are transcribed into heterogenous nuclearRNA (hnRNA); introns may contain regulatory elements such as enhancers.Introns are removed or “spliced out” from the nuclear or primarytranscript;

[0121] introns therefore are absent in the messenger RNA (mRNA)transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide.

[0122] In addition to containing introns, genomic forms of a gene mayalso include sequences located on both the 5′ and 3′ end of thesequences which are present on the RNA transcript. These sequences arereferred to as “flanking” sequences or regions (these flanking sequencesare located 5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

[0123] A “transformed cell” is a cell or cell line that has acquired theability to grow in cell culture for many multiple generations, theability to grow in soft agar and the ability to not have cell growthinhibited by cell-to-cell contact. In this regard, transformation refersto the introduction of foreign genetic material into a cell or organism.Transformation may be accomplished by any method known which permits thesuccessful introduction of nucleic acids into cells and which results inthe expression of the introduced nucleic acid. “Transformation” includesbut is not limited to such methods as transfection, microinjection,electroporation, and lipofection (liposome-mediated gene transfer).Transformation may be accomplished through use of any expression vector.For example, the use of baculovirus to introduce foreign nucleic acidinto insect cells is contemplated. The term “transformation” alsoincludes methods such as P-element mediated germline transformation ofwhole insects. Additionally, transformation refers to cells that havebeen transformed naturally, usually through genetic mutation.

[0124] As used herein, the term “in vitro” refers to an artificialenvironment and to processes or reactions that occur within anartificial environment. In vitro environments can consist of, but arenot limited to, test tubes and cell culture. The term “in vivo” refersto the natural environment (e.g., an animal or a cell) and to processesor reaction that occur within a natural environment.

[0125] The terms “test compound” and “candidate compound” refer to anychemical entity, pharmaceutical, drug, and the like that is a candidatefor use to treat or prevent a disease, illness, sickness, or disorder ofbodily function (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. In some embodiments of the present invention, test compoundsinclude antisense compounds.

[0126] As used herein, the term “sample” is used in its broadest sense.In one sense, it is meant to include a specimen or culture obtained fromany source, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

[0127] Experimental

[0128] The following examples are provided in order to demonstrate andfurther illustrate certain preferred embodiments and aspects of thepresent invention and are not to be construed as limiting the scopethereof In the experimental disclosure which follows, the followingabbreviations apply: ° C. (degrees Centigrade); rpm (revolutions perminute); BSA (bovine serum albumin); CFA (complete Freund's adjuvant);IFA (incomplete Freund's adjuvant); IgG (immunoglobulin G); IM(intramuscular); IP (intraperitoneal); IV (intravenous orintravascular); SC (subcutaneous); H₂O (water); HCl (hydrochloric acid);aa (amino acid); bp (base pair); kb (kilobase pair); kD (kilodaltons);gm (grams); μg (micrograms); mg (milligrams); ng (nanograms); μl(microliters); ml (milliliters); mm (millimeters); nm (nanometers); μm(micrometer); M (molar); mM (millimolar); μM (micromolar); U (units); V(volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes);hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl (sodium chloride);OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (optical density at 600 nm);PAGE (polyacrylamide gel electrophoresis); PBS (phosphate bufferedsaline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PCR(polymerase chain reaction); PEG (polyethylene glycol); PMSF(phenylmethylsulfonyl fluoride); RT-PCR (reverse transcription PCR); SDS(sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); w/v(weight to volume); v/v (volume to volume); BAC (bacterial artificialchromosome); and YAC (yeast artificial chromosome).

[0129] Materials and equipment were obtained from the following sources:Ambion (Ambion, Inc., Austin, Tex.); Amersham (Amersham PharmaciaBiotechnology, Inc., Piscataway, N.J.); Amicon (Amicon, Inc., Beverly,Mass.); ATCC (American Type Culture Collection, Rockville, Md.); AppliedSpectral Imaging (Applied Spectral Imaging, Carlsbad, Calif.); BectonDickinson (Becton Dickinson Labware, Lincoln Park, N.J.); BioRad(BioRad, Richmond, Calif.); Chroma (Chroma Technology, Brattleboro,Vt.); Clontech (Clontech Laboratories, Palo Alto, Calif.); Difco (DifcoLaboratories, Detroit, Mich.); GIBCO BRL or Gibco BRL (LifeTechnologies, Inc., Gaithersburg, Md.); HyClone (HyClone, Logan, Utah);ICN (ICN Pharmaceuticals, Inc., Costa Mesa, Calif.); Invitrogen(Invitrogen Corp., San Diego, Calif.); Leica Microsystems (LeicaMicrosystems Imaging Solutions, Cambridge, United Kingdom); Leica(Leica, Wetzlar, Germany); Molecular Probes (Molecular Probes, Eugene,Oreg.); New England Biolabs (New England Biolabs, Inc., Beverly, Mass.);Novagen (Novagen, Inc., Madison, Wis.); Oakland BAC/PAC Resources(Oakland BAC/PAC Resources, Oakland, Calif.); Perkin Elmer or PE (PerkinElmer Applied Biosystems, Foster City, Calif.); Photometrics(Photometrics, Tucson, Ariz.); Promega (Promega Corporation, Madison,Wis.); Qiagen (Qiagen Inc., Valencia, Calif.); Research Genetics(Research Genetics, Huntsville, Ala.); Roche (Hoffmann La Roche,Indianapolis, Ind.); Sigma Aldrich (Sigma Aldrich Chemical Co., St.Louis, Mo.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.);Vector (Vector Laboratories, Burlingame, Calif.); and Vysis (Vysis,Downers Grove, Ill.).

EXAMPLE 1 Tumor Samples

[0130] In this Example, the tumor samples used during the development ofthe present invention are described. The H292 and H3118 (pulmonary MEC),the H727 (pulmonary carcinoid), and H2009 (non-small cell lung cancer)tumor cell lines were generated from patient biopsy samples at theNational Naval Medical Center as known in the art (See, Carney et al.,Cancer Res., 45:2913-23 [1985]; and Modi et al., Oncogene 19:4632-9[2000]) using an IRB-approved tissue procurement protocol. The pulmonaryMEC tumor sample (MECT #A) was obtained as an anonymous tumor sampleapproved for inclusion in this study by the NIH Office of Human SubjectsResearch. Human U2OS osteosarcoma cells were cultured in Dulbecco'smodified Eagle's medium (DMEM) containing 10% Fetalclone I serum(HyClone). COS7 cells were cultured in RPMI 1640 medium supplementedwith 10% FCS. NIH 3T3 cell transduced with the pBABE retrovirus encodingJagged 2, or empty pBABE retrovirus, were maintained in DMEM containing10% FCS and 1 mg/ml puromycin.

EXAMPLE 2 Spectral Karyotyping

[0131] In this Example, the spectral karyotyping methods used in thedevelopment of the present invention are briefly described (See, Tononet al., Genes Chromosomes Cancer 27:418-23 [2000]). Specificchromosomes, kindly provided by Dr. Thomas Ried, were obtained byhigh-resolution flow sorting, and then amplified using two consecutiverounds of degenerate oligo-primed (DOP)-PCR amplification. Methodscommonly used and widely known in the art were used for the spectralkaryotyping experiments.

[0132] Spectrum Orange (Vysis), Rhodamine 110 (Perkin Elmer), and TexasRed (Molecular Probes) were used for the direct labeling of chromosomes,whereas Biotin-16-dUTP and Digoxigenin-11-DUTP (Roche) were used for theindirect labeling of chromosomes. After hybridization, biotin wasdetected with Avidin-Cy5 (Amersham) and digoxigenin-11-dUTP with mouseanti-digoxin (Sigma) followed by sheep anti-mouse antibodiescustom-conjugated to Cy5.5 (Amersham). The slides were counterstainedwith 4,6-diamidino-2-phenylindole (DAPI, Sigma) and covered withantifade solution (Vector). Spectral images were acquired with an SD200SpectraCube system (Applied Spectral Imaging) mounted on a Leica DMRBEmicroscope (Leica) through a custom-designed triple bandpass opticalfilter (SKY v.3; Chroma). Spectrum-based classification of the rawspectral images was performed using SKYView 1.6 software (AppliedSpectral Imaging).

EXAMPLE 3 Fluorescence in Situ Hybridization (FISH) Analysis

[0133] In this Example, the FISH analysis used during the development ofthe present invention is described. BAC clones were purchased fromResearch Genetics, Oakland BAC/PAC Resources, or provided by Dr. RalucaJonescu (RP11-16K5). For the FISH analysis, BAC clones were labeled bynick translation. BAC clones RP11-676L3 and RP11-16K5 were used toidentify translocations. Image acquisition was performed using a SensysCCD camera (Photometrics), and Q-FISH software (Leica MicrosystemsImaging Solutions). Using standard FISH protocols, specifictranslocation events were detected. Additional experiments to identifytranslocation using a single probe were also conducted using methodsknown in the art.

EXAMPLE 4 Nucleic Acid Analysis

[0134] In this Example, nucleic acid analysis methods used during thedevelopment of the present invention are described. Total RNA wasobtained from tumor samples using guanidine isothiocyanate methodologyas known in the art (See, Sambrook et al., Molecular cloning: alaboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.[1989]), and subjected to 5′ and 3′ RACE using conditions as recommendedby the manufacturer (SmartRace, Clontech). RT-PCR with gene-specificoligonucleotides for MECT/MAML2 was performed as recommended by themanufacturer (Amersham Pharmacia). The method utilized first-strand cDNAfrom oligo-dT primers, followed by PCR using gene-specificoligonucleotides. The PCR conditions included a denaturation step at 95°C. for 30 seconds, annealing at 55° C. for 30 seconds, and extension at72° C. for 30 seconds.

[0135] In these methods, the oligonucleotide primers used to detectspecific MECT1/MAML2 fusion mRNA in mucoepidermoid tumors had thefollowing sequences: MECT1 Exon 1 Sense, 5′-CGA GAA GAT GGC GAC TTC GAACA-3′ (SEQ ID NO:10) and MAML2 Exon 2 Antisense, 5′-CCA TTG GGT CGC TTGCTG TTG GCA GGA G-3′ (SEQ ID NO:11). RT-PCR produced a distinct 203 bpsignal in all mucoepidermoid tumor samples tested. However, this signalwas not detected in other tumor types tested. It is contemplated thatthis signal is detectable in all tumor samples carrying the t(11;19)translocation described herein.

[0136] The MAML2, MECT1-MAML2, and MAML2 (172-1153) cDNAs were subclonedinto a CMV-2 driven expression vector in-frame with the sequenceencoding the FLAG tag (pFLAG-CMV2) and into the pEGFP-C3 (Clontech) andpBIND (Promega) vectors. All constructs were confirmed by nucleotidesequencing and immunoblotting. The full-length MECT1-MAML2 was cloned,as a Sal I-Not I fragment, into pEGFP-C3 and pBIND. HA-epitope-taggedICN1 and myc-epitope tagged CSL have been previously described (Wu etal., supra [2000]). HES1-luc contains the −194 to +160 promoter fragmentof the HES1 gene cloned upstream of the firefly luciferase gene in thepGL2-basic vector (Jarriault et al., Nature 377:355-358 [1995]), andHES1-Δ-luc, derived from HES1-luc, has a deletion removing the two CSLbinding sites. hTERT-luc was obtained by cloning 2.5 kb of the hTERTpromoter (Greenberg et al., Oncogene 18:1219-1226 [1999]) intopGL3-basic vector. p21-luc (Tang et al., J. Biol. Chem., 273:29156-29163[1998]), p27-luc (Kwon et al., Gene 180:113-120 [1996]), HES-5-luc(Beatus et al., Development 126:3925-3935 [1999]) and HES-7 (Bessho etal., Genes Cell 6:175-185 [2001]) have been previously described. pRL-TK(Promega) which contains a Renilla luciferase insert under control ofthe thymidine kinase promoter, was used to normalize firefly luciferaseactivity in order to determine transfection efficiency. pSG5-luc(Promega) is a firefly luciferase reporter plasmid that contains fivecopies of the GAL4 binding site upstream of a minimal TATA box.

EXAMPLE 5 Protein Studies

[0137] This Example describes the materials and methods usedimmunofluorescence analysis and immunoprecipitation. The followingantibodies were obtained from commercial sources: mouse anti-Flagantibody (clone M2, Sigma); mouse anti-HA antibody (clone HA. 11,Babco); mouse anti-Myc antibody (clone 9E10, Clontech); horseradishperoxidase (HRP)-coupled goat anti-mouse antibody (Amersham).Transfections were carried out using Superfect transfection reagent(QIAGEN) according to the manufacturer's instructions. At 48 hrspost-transfection, cells were washed with ice-cold PBS and lysed in situwith a solution containing 20 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40(w/v), 10% glycerol (w/v), 100 mM NaF, 1 mM phenylmethylsulfonylfluoride (PMSF), 20 μg/ml aprotinin, 1 mM sodium orthovanadate (Na₃VO₄),and 40 μg/ml leupeptin. After incubation on ice for 30 min, cell lysateswere centrifuged at 12,000 g for 15 min at 4° C. Cleared lysates wereincubated with anti-Flag antibody (M2 at 1:500) and anti-mouse IgGagarose (Sigma) for 4 h or overnight at 4° C. The washed pellets werethen subjected to SDS-PAGE and western blotting using the indicatedantibodies. Washed membranes were incubated with HRP-coupled secondaryantibodies for 1 hr, washed again and stained using a chemiluminescentreagent (ECL, Amersham).

EXAMPLE 6 Luciferase Assays

[0138] In this Example, the materials and methods for analysis oftranscription are described. Briefly, cells were seeded on the six-wellplates at 1×10⁵ cells per well (U20S and HeLa cells), or at 2×10⁵ cellsper well (293 cells) 1 day prior to transient co-transfection using theindicated plasmid DNA combinations and concentrations. In theseexperiments, the total plasmids concentration was kept constant byadding appropriate quantities of vectors without inserts. Transfectedcells were harvested 48 hrs post-transfection and luciferase activitieswere measured in a Berthold luminometer (Lumat LB9507) using the dualluciferase reporter assay system (Promega) as directed by themanufacturer. Relative luciferase activities were normalized to Renillaluciferase activity.

[0139] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the relevant fields are intended to be within the scopeof the following claims.

1 12 1 1153 PRT Homo sapiens 1 Met Gly Asp Thr Ala Pro Pro Gln Ala ProAla Gly Gly Leu Gly Gly 1 5 10 15 Ala Ser Gly Ala Gly Leu Leu Gly GlyGly Ser Val Thr Pro Arg Val 20 25 30 His Ser Ala Ile Val Glu Arg Leu ArgAla Arg Ile Ala Val Cys Arg 35 40 45 Gln His His Leu Ser Cys Glu Gly ArgTyr Glu Arg Gly Arg Ala Glu 50 55 60 Ser Ser Asp Arg Glu Arg Glu Ser ThrLeu Gln Leu Leu Ser Leu Val 65 70 75 80 Gln His Gly Gln Gly Ala Arg LysAla Gly Lys His Thr Lys Ala Thr 85 90 95 Ala Thr Ala Ala Thr Thr Thr AlaPro Pro Pro Pro Pro Ala Ala Pro 100 105 110 Pro Ala Ala Ser Gln Ala AlaAla Thr Ala Ala Pro Pro Pro Pro Pro 115 120 125 Asp Tyr His His His HisGln Gln His Leu Leu Asn Ser Ser Asn Asn 130 135 140 Gly Gly Ser Gly GlyIle Asn Gly Glu Gln Gln Pro Pro Ala Ser Thr 145 150 155 160 Pro Gly AspGln Arg Asn Ser Ala Leu Ile Ala Leu Gln Gly Ser Leu 165 170 175 Lys ArgLys Gln Val Val Asn Leu Ser Pro Ala Asn Ser Lys Arg Pro 180 185 190 AsnGly Phe Val Asp Asn Ser Phe Leu Asp Ile Lys Arg Ile Arg Val 195 200 205Gly Glu Asn Leu Ser Ala Gly Gln Gly Gly Leu Gln Ile Asn Asn Gly 210 215220 Gln Ser Gln Ile Met Ser Gly Thr Leu Pro Met Ser Gln Ala Pro Leu 225230 235 240 Arg Lys Thr Asn Thr Leu Pro Ser His Thr His Ser Pro Gly AsnGly 245 250 255 Leu Phe Asn Met Gly Leu Lys Glu Val Lys Lys Glu Pro GlyGlu Thr 260 265 270 Leu Ser Cys Ser Lys His Met Asp Gly Gln Met Thr GlnGlu Asn Ile 275 280 285 Phe Pro Asn Arg Tyr Gly Asp Asp Pro Gly Glu GlnLeu Met Asp Pro 290 295 300 Glu Leu Gln Glu Leu Phe Asn Glu Leu Thr AsnIle Ser Val Pro Pro 305 310 315 320 Met Ser Asp Leu Glu Leu Glu Asn MetIle Asn Ala Thr Ile Lys Gln 325 330 335 Asp Asp Pro Phe Asn Ile Asp LeuGly Gln Gln Ser Gln Arg Ser Thr 340 345 350 Pro Arg Pro Ser Leu Pro MetGlu Lys Ile Val Ile Lys Ser Glu Tyr 355 360 365 Ser Pro Gly Leu Thr GlnGly Pro Ser Gly Ser Pro Gln Leu Arg Pro 370 375 380 Pro Ser Ala Gly ProAla Phe Ser Met Ala Asn Ser Ala Leu Ser Thr 385 390 395 400 Ser Ser ProIle Pro Ser Val Pro Gln Ser Gln Ala Gln Pro Gln Thr 405 410 415 Gly SerGly Ala Ser Arg Ala Leu Pro Ser Trp Gln Glu Val Ser His 420 425 430 AlaGln Gln Leu Lys Gln Ile Ala Ala Asn Arg Gln Gln His Ala Arg 435 440 445Met Gln Gln His Gln Gln Gln His Gln Pro Thr Asn Trp Ser Ala Leu 450 455460 Pro Ser Ser Ala Gly Pro Ser Pro Gly Pro Phe Gly Gln Glu Lys Ile 465470 475 480 Pro Ser Pro Ser Phe Gly Gln Gln Thr Phe Ser Pro Gln Ser SerPro 485 490 495 Met Pro Gly Val Ala Gly Gly Ser Gly Gln Ser Lys Val MetAla Asn 500 505 510 Tyr Met Tyr Lys Ala Gly Pro Ser Ala Gln Gly Gly HisLeu Asp Val 515 520 525 Leu Met Gln Gln Lys Pro Gln Asp Leu Ser Arg SerPhe Ile Asn Asn 530 535 540 Pro His Pro Ala Met Glu Pro Arg Gln Gly AsnThr Lys Pro Leu Phe 545 550 555 560 His Phe Asn Ser Asp Gln Ala Asn GlnGln Met Pro Ser Val Leu Pro 565 570 575 Ser Gln Asn Lys Pro Ser Leu LeuHis Tyr Thr Gln Gln Gln Gln Gln 580 585 590 Gln Gln Gln Gln Gln Gln GlnGln Gln Gln Gln Gln Gln Gln Gln Gln 595 600 605 Gln Gln Gln Gln Gln GlnGln Gln Gln Gln Ser Ser Ile Ser Ala Gln 610 615 620 Gln Gln Gln Gln GlnGln Ser Ser Ile Ser Ala Gln Gln Gln Gln Gln 625 630 635 640 Gln Gln GlnGln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 645 650 655 Gln GlnGln Gln Gln Gln Pro Ser Ser Gln Pro Ala Gln Ser Leu Pro 660 665 670 SerGln Pro Leu Leu Arg Ser Pro Leu Pro Leu Gln Gln Lys Leu Leu 675 680 685Leu Gln Gln Met Gln Asn Gln Pro Ile Ala Gly Met Gly Tyr Gln Val 690 695700 Ser Gln Gln Gln Arg Gln Asp Gln His Ser Val Val Gly Gln Asn Thr 705710 715 720 Gly Pro Ser Pro Ser Pro Asn Pro Cys Ser Asn Pro Asn Thr GlySer 725 730 735 Gly Tyr Met Asn Ser Gln Gln Ser Leu Leu Asn Gln Gln LeuMet Gly 740 745 750 Lys Lys Gln Thr Leu Gln Arg Gln Ile Met Glu Gln LysGln Gln Leu 755 760 765 Leu Leu Gln Gln Gln Met Leu Ala Asp Ala Glu LysIle Ala Pro Gln 770 775 780 Asp Gln Ile Asn Arg His Leu Ser Arg Pro ProPro Asp Tyr Lys Asp 785 790 795 800 Gln Arg Arg Asn Val Gly Asn Met GlnPro Thr Ala Gln Tyr Ser Gly 805 810 815 Gly Ser Ser Thr Ile Ser Leu AsnSer Asn Gln Ala Leu Ala Asn Pro 820 825 830 Val Ser Thr His Thr Ile LeuThr Pro Asn Ser Ser Leu Leu Ser Thr 835 840 845 Ser His Gly Thr Arg MetPro Ser Leu Ser Thr Ala Val Gln Asn Met 850 855 860 Gly Met Tyr Gly AsnLeu Pro Cys Asn Gln Pro Asn Thr Tyr Ser Val 865 870 875 880 Thr Ser GlyMet Asn Gln Leu Thr Gln Gln Arg Asn Pro Lys Gln Leu 885 890 895 Leu AlaAsn Gln Asn Asn Pro Met Met Pro Arg Pro Pro Thr Leu Gly 900 905 910 ProSer Asn Asn Asn Asn Val Ala Thr Phe Gly Ala Gly Ser Val Gly 915 920 925Asn Ser Gln Gln Leu Arg Pro Asn Leu Thr His Ser Met Ala Ser Met 930 935940 Pro Pro Gln Arg Thr Ser Asn Val Met Ile Thr Ser Asn Thr Thr Ala 945950 955 960 Pro Asn Trp Ala Ser Gln Glu Gly Thr Ser Lys Gln Gln Glu AlaLeu 965 970 975 Thr Ser Ala Gly Val Arg Phe Pro Thr Gly Thr Pro Ala AlaTyr Thr 980 985 990 Pro Asn Gln Ser Leu Gln Gln Ala Val Gly Ser Gln GlnPhe Ser Gln 995 1000 1005 Arg Ala Val Ala Pro Pro Asn Gln Leu Thr ProAla Val Gln Met 1010 1015 1020 Arg Pro Met Asn Gln Met Ser Gln Thr LeuAsn Gly Gln Thr Met 1025 1030 1035 Gly Pro Leu Arg Gly Leu Asn Leu ArgPro Asn Gln Leu Ser Thr 1040 1045 1050 Gln Ile Leu Pro Asn Leu Asn GlnSer Gly Thr Gly Leu Asn Gln 1055 1060 1065 Ser Arg Thr Gly Ile Asn GlnPro Pro Ser Leu Thr Pro Ser Asn 1070 1075 1080 Phe Pro Ser Pro Asn GlnSer Ser Arg Ala Phe Gln Gly Thr Asp 1085 1090 1095 His Ser Ser Asp LeuAla Phe Asp Phe Leu Ser Gln Gln Asn Asp 1100 1105 1110 Asn Met Gly ProAla Leu Asn Ser Asp Ala Asp Phe Ile Asp Ser 1115 1120 1125 Leu Leu LysThr Glu Pro Gly Asn Asp Asp Trp Met Lys Asp Ile 1130 1135 1140 Asn LeuAsp Glu Ile Leu Gly Asn Asn Ser 1145 1150 2 593 PRT Homo sapiens 2 MetAla Thr Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys Ile Ala Leu 1 5 10 15His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe Glu Glu Val Met 20 25 30Lys Asp Leu Ser Leu Thr Arg Ala Ala Arg Leu Gln Leu Gln Lys Ser 35 40 45Gln Tyr Leu Gln Leu Gly Pro Ser Arg Gly Gln Tyr Tyr Gly Gly Ser 50 55 60Leu Pro Asn Val Asn Gln Ile Gly Ser Gly Thr Met Asp Leu Pro Phe 65 70 7580 Gln Thr Pro Phe Gln Ser Ser Gly Leu Asp Thr Ser Arg Thr Thr Arg 85 9095 His His Gly Leu Val Asp Arg Val Tyr Arg Glu Arg Gly Arg Leu Gly 100105 110 Ser Pro His Arg Arg Pro Leu Ser Val Asp Lys His Gly Arg Gln Ala115 120 125 Asp Ser Cys Pro Tyr Gly Thr Met Tyr Leu Ser Pro Pro Ala AspThr 130 135 140 Ser Trp Arg Arg Thr Asn Ser Asp Ser Ala Leu His Gln SerThr Met 145 150 155 160 Thr Pro Thr Gln Pro Glu Ser Phe Ser Ser Gly SerGln Asp Val His 165 170 175 Gln Lys Arg Val Leu Leu Leu Thr Val Pro GlyMet Glu Glu Thr Thr 180 185 190 Ser Glu Ala Asp Lys Asn Leu Ser Lys GlnAla Trp Asp Thr Lys Lys 195 200 205 Thr Gly Ser Arg Pro Lys Ser Cys GluVal Pro Gly Ile Asn Ile Phe 210 215 220 Pro Ser Ala Asp Gln Glu Asn ThrThr Ala Leu Ile Pro Ala Thr His 225 230 235 240 Asn Thr Gly Gly Ser LeuPro Asp Leu Thr Asn Ile His Phe Pro Ser 245 250 255 Pro Leu Pro Thr ProLeu Asp Pro Glu Glu Pro Thr Phe Pro Ala Leu 260 265 270 Ser Ser Ser SerSer Thr Gly Asn Leu Ala Ala Asn Leu Thr His Leu 275 280 285 Gly Ile GlyGly Ala Gly Gln Gly Met Ser Thr Pro Gly Ser Ser Pro 290 295 300 Gln HisArg Pro Ala Gly Val Ser Pro Leu Ser Leu Ser Thr Glu Ala 305 310 315 320Arg Arg Gln Gln Ala Ser Pro Thr Leu Ser Pro Leu Ser Pro Ile Thr 325 330335 Gln Ala Val Ala Met Asp Ala Leu Ser Leu Glu Gln Gln Leu Pro Tyr 340345 350 Ala Phe Phe Thr Gln Ala Gly Ser Gln Gln Pro Pro Pro Gln Pro Gln355 360 365 Pro Pro Pro Pro Pro Pro Pro Ala Ser Gln Gln Pro Pro Pro ProPro 370 375 380 Pro Pro Gln Ala Pro Val Arg Leu Pro Pro Gly Gly Pro LeuLeu Pro 385 390 395 400 Ser Ala Ser Leu Thr Arg Gly Pro Gln Pro Pro ProLeu Ala Val Thr 405 410 415 Val Pro Ser Ser Leu Pro Gln Ser Pro Pro GluAsn Pro Gly Gln Pro 420 425 430 Ser Met Gly Ile Asp Ile Ala Ser Ala ProAla Leu Gln Gln Tyr Arg 435 440 445 Thr Ser Ala Gly Ser Pro Ala Asn GlnSer Pro Thr Ser Pro Val Ser 450 455 460 Asn Gln Gly Phe Ser Pro Gly SerSer Pro Gln Leu Glu Gln Phe Asn 465 470 475 480 Met Met Glu Asn Ala IleSer Ser Ser Ser Leu Tyr Ser Pro Gly Ser 485 490 495 Thr Leu Asn Tyr SerGln Ala Ala Met Met Gly Leu Thr Gly Ser His 500 505 510 Gly Ser Leu ProAsp Ser Gln Gln Leu Gly Tyr Ala Ser His Ser Gly 515 520 525 Ile Pro AsnIle Ile Leu Thr Val Thr Gly Glu Ser Pro Pro Ser Leu 530 535 540 Ser LysGlu Leu Thr Ser His Arg Gly His Leu Pro Asp Gly Pro Pro 545 550 555 560Val Ser Gly His Ala Gly Thr Leu Pro Leu Ser Arg Pro Asp Gly Ala 565 570575 Ser Pro Ala Arg Gly Arg Pro Cys Ser Val Pro Arg Gln Arg Pro Ser 580585 590 Leu 3 5419 DNA Homo sapiens 3 ctccctctcc tatcggagca caatgaaagcctgtgtatcg ccgtgactcc gggcgggagc 60 cagtgtcagc aaagcggcta acaacagacgagaaagagaa aggaaaatac aagctacttt 120 ttttttccat ctataaagcg gagcaaatacaggagataga accagattgc ttattgcgag 180 tccagaccct cagatccact ggccggggatggaatgtaca aaagtggaca gaaaagtggc 240 tggacatgac tcggtgcaat ttgctggaagtttgtaagtt tgaccatcgt ttgtaaatta 300 ctctcggaag agtttgtctc tcttgatactgtattagaat agagccgggg gtgaggaata 360 gaaacgtaag cgggaaagaa aaaaatgtgttgaaggatct ctctcagtgg ctagcgactt 420 aagattgctt ttcatttaag gctaggaaaccttagaggga gtgaggattt taccggtgat 480 tggattagct gaagaaaaaa gcatggtccaaaagtccaat tactgacatt gttaacagtt 540 gaaaagctgt ctccctcttt tgggagaagacaacatccta cagtacccca aagaggagaa 600 aacaccggag cgaaaggaaa gggaggaaaaattaaaagcc aaaagacagt ctcccttgat 660 ttttgcacat tttgaacagt gacttaaacatcttctgaaa cagcactgtt ttgttttgtt 720 ttggtttttt atttaacctg aggaaaagtcaaggctgctg gttacataga catggtagaa 780 atgtgtttct ctgcagaaac atccccataaagaattgtcg gaaacaacta ggtgaggggg 840 agtcctctct attaatacct ctctcaataccttttgctgt gtgtttctgt ctcttgctgg 900 acaatccctg aattcttgat ctaacccccagatcgtgtgt ttacaaagta cctagtggct 960 cttgtcagct tggtggagga aaaaaaatccaccaactctg tccaacttct ccagagctgt 1020 caaatgcaat tagagtaagt taatcagggtttgtttccaa cttatcctcc ccccagttgg 1080 tttctattct ttctccccac cctctttttactaactcccc tcccccacaa cttctccacg 1140 gctcccccac aacctctgaa gacctctattcatgtggccc tgaacactga gctcacattg 1200 tcaaaaacag acttgcctgc aatagccagcagtagcctct ttccacctca ccatcccaga 1260 ggcagcaatc attgtgtccg gtaagatgggggacacagcg cccccgcagg cccccgcagg 1320 agggctaggg ggggcctctg gggcggggctccttggaggg ggctcagtca ccccgagagt 1380 gcacagtgct atcgtggagc gcctccgggctcggatcgct gtctgccgcc aacaccacct 1440 gagctgtgaa ggacgatatg aacgaggtagggccgagagc tcagaccggg aaagagaaag 1500 caccttgcag ctcctgagcc ttgtacagcatggccagggg gcaaggaaag ctggcaaaca 1560 caccaaggcc accgccactg ctgccaccactacagcccct ccaccgcccc ctgctgcccc 1620 tcctgcggcc tcccaagcag cagcaacagcagccccaccg cccccaccag actatcacca 1680 tcaccaccag cagcacctgc tgaacagtagcaataatggt ggcagtggtg ggataaacgg 1740 agagcagcag ccgcccgctt caaccccaggggaccagagg aactcagccc tgattgcgct 1800 ccagggttcc ttgaaaagaa aacaggtagttaacctatct cctgccaaca gcaagcgacc 1860 caatggcttt gtggacaact catttcttgatatcaaaaga attcgtgttg gggagaatct 1920 ctctgcagga caaggtggcc tccaaataaacaatggacaa agtcagatta tgtcagggac 1980 cttgcctatg agccaagcac ccctgcgaaagactaacact ctgccatccc atacacattc 2040 tcctggcaat ggcctgttta acatgggcttaaaggaggta aagaaggagc caggagagac 2100 tctgtcttgc agtaagcaca tggatggccaaatgacccaa gagaatattt ttcctaatag 2160 gtacggagac gaccctggag aacaactgatggatcctgag ctgcaggaac tgttcaatga 2220 actgaccaac atatctgtgc ctcccatgagtgaccttgaa ctggagaaca tgatcaatgc 2280 caccataaag caggatgacc catttaacattgacttgggt cagcaaagcc agaggagcac 2340 acctaggccc tccttaccca tggagaaaatagtgatcaaa agtgaatact caccgggctt 2400 gactcagggc ccctcaggct ctcctcagctgaggccccca tcagctggcc ccgcattctc 2460 catggccaac tctgccctct ccacttcgtctccaatccct tcagtccctc agagccaggc 2520 tcagcctcag acaggctccg gagcaagccgggccttgcca agctggcagg aagtatccca 2580 tgcccagcag ctcaaacaga tagctgctaatcgtcagcag catgcccgga tgcagcagca 2640 ccagcagcag caccagccta ccaactggtcagccttgccc tcctctgctg gaccatcacc 2700 aggtccattt gggcaggaga aaatccccagcccttctttt ggtcagcaga cattcagccc 2760 acagagctcc cccatgcctg gggtagctggcggcagcggc cagtcgaaag taatggctaa 2820 ctacatgtac aaggccggcc cctcagcccagggtgggcac ctagatgtcc tcatgcagca 2880 aaagcctcag gatctcagtc gaagttttattaacaacccg cacccagcca tggagccccg 2940 tcagggcaac accaagcctt tgtttcattttaactcagat caagcgaacc agcagatgcc 3000 ttctgttttg ccttcccaga acaagccttctctcctacac tacacccaac agcaacagca 3060 gcaacagcag cagcagcagc agcagcagcagcagcaacag cagcagcagc agcaacagca 3120 acagcaacag caacagcaga gttcaatttcagctcaacaa cagcaacagc agcagagctc 3180 aatttcagcc caacagcagc agcagcagcaacaacagcag cagcagcagc aacaacaaca 3240 gcaacaacag cagcagcagc agcagcaacaaccatcttct cagcctgccc aatctctacc 3300 aagccagcct ttgctaaggt cacctttgccacttcagcaa aagctcctac ttcagcaaat 3360 gcagaatcag cccattgcag gaatgggataccaagtctcc caacaacaga gacaggatca 3420 acactctgtg gtaggccaga acacaggccccagtccaagt cctaacccct gctcaaatcc 3480 aaacactgga agtggttaca tgaactcccagcaatcactg ttgaatcagc aattgatggg 3540 aaagaagcag actctacaga ggcagatcatggagcagaaa cagcaacttc ttctccagca 3600 gcagatgctg gctgacgcgg agaaaattgctccacaagat cagataaacc gacatttgtc 3660 aaggccacct ccagattata aagaccaaagaagaaatgtg ggcaatatgc aaccaactgc 3720 tcagtattct ggtggctcat ccacaataagcttaaactct aaccaggctt tggcaaaccc 3780 agtttcaaca cacaccattt taactcccaattccagcctc ctgtctactt ctcacgggac 3840 aagaatgcca tcattatcta cagcagttcagaatatgggg atgtatggaa atctgccttg 3900 taatcaacct aacacataca gtgtcacttcaggaatgaat caattgaccc aacagagaaa 3960 cccaaagcaa ttgttagcaa atcaaaacaaccctatgatg ccacggccac ctaccttagg 4020 gccaagtaat aataacaatg tagccacttttggagctgga tctgttggta attcacaaca 4080 attgagacca aatttaaccc atagtatggcaagcatgcca ccacagagaa catcaaacgt 4140 aatgatcaca tccaacacaa ctgcaccaaactgggcctct caagaaggaa caagcaaaca 4200 gcaagaagcc ctgacgtctg caggagtccgcttccccaca ggtacacctg cagcctatac 4260 cccaaatcag tcactgcaac aggcagtaggtagccagcaa ttttcccaga gggcagtggc 4320 tcctcctaac cagttaacac cagcagtgcaaatgagaccc atgaaccaaa tgagccaaac 4380 actaaatggg caaaccatgg gtcccctcaggggtctgaat ctcagaccca atcagctaag 4440 cacacagatt ttgcctaatt tgaatcagtcaggaacaggg ttgaatcagt cgaggacggg 4500 catcaaccag ccaccatccc tgacgcccagcaattttcct tcacccaacc aaagttccag 4560 ggcttttcaa ggaactgacc acagcagtgacttagctttt gacttcctca gccaacaaaa 4620 tgataacatg ggccctgccc taaacagtgatgctgatttc attgattctt tattgaagac 4680 agagcctggt aatgatgact ggatgaaagacatcaatctt gatgaaatct tggggaacaa 4740 ttcctaaaga agaaagggaa gacaatttacaaactccaag cactaaaagg cagtatatta 4800 cagaaactct gtagaggctg aactgttgatgttcaggtgg actacatgaa gataacatgc 4860 ttaaaaatgg aaagcagaaa gtaactgcagtgatgaacat tttggtccaa attcttgttt 4920 taaatcttac acctgaaagt aaaatattgggatcactttt ccctgtctaa actccaggat 4980 acagtatcca atttatccaa acagaactgtggtgtcaatg tgtaattaat tgtgtaaaat 5040 agccttccca agtttctttt tccctggaaaataaaaaagg taatagaact tgtagtttat 5100 ttaaacccca tgtcatgagg aggtactagttccaagcaac aaactcctta atttgctcta 5160 atagataggt atggtttaat ctttccattgtgtcttttca tttaattttc ctgaagcttg 5220 caggatagat tgaaatgtta taggtttgtttggagtaacc aaacagtatg caaattaaga 5280 aaaagccaga gaacctagaa aacatccagtggattacaga atttcttccc catattcact 5340 cctcactttt acaattttcc cacaatcctctacttcagtg ggatgctgtg tctagtgatt 5400 aaacaaaaat atagagctg 5419 4 2342DNA Homo sapiens 4 ccagccggcg cttgcgcggt ggcacgggcg agtgggggggcgaggaggtg gaggaggagg 60 aggaggagga ggaggtggcg gcgagaagat ggcgacttcgaacaatccgc ggaaattcag 120 cgagaagatc gcgctgcaca atcagaagca ggcggaggagacggcggcct tcgaggaggt 180 catgaaggac ctgagcctga cgcgggccgc gcggctccagctccagaaat cccagtacct 240 gcaactgggc cccagccgag gccagtacta tggcgggtccctgcccaacg tgaaccagat 300 cgggagtggc accatggacc tgcccttcca gaccccctcccaatcctcgg gcctggacac 360 cagccggacc acccggcacc atgggctggt ggacagggtgtaccgggagc gtggccggct 420 cggctcccca caccgccggc ccctgtcagt ggacaaacacggacggcagg ccgacagctg 480 cccctatggc accatgtacc tctcaccacc cgcggacaccagctggagaa ggaccaattc 540 tgactccgcc ctgcaccaga gcacaatgac gcccacgcagccagaatcct ttagcagtgg 600 gtcccaggac gtgcaccaga aaagagtctt actgttaacagtcccaggaa tggaagagac 660 cacatcagag gcagacaaaa acctttccaa gcaagcatgggacaccaaga agacggggtc 720 caggcccaag tcctgtgagg tccccggaat caacatcttcccgtctgccg accaggaaaa 780 cactacagcc ctgatccccg ccacccacaa cacaggggggtccctgcccg acctgaccaa 840 catccacttc ccctccccgc tcccgacccc gctggaccccgaggagccca ccttccctgc 900 actgagcagc tccagcagca ccggcaacct cgcggccaacctgacgcacc tgggcatcgg 960 tggcgccggc cagggaatga gcacacctgg ctcctctccacagcaccgcc cagctggcgt 1020 cagccccctg tccctgagca cagaggcaag gcgtcagcaggcatcgccca ccctgtcccc 1080 gctgtcaccc atcactcagg ctgtagccat ggacgccctgtctctggagc agcagctgcc 1140 ctacgccttc ttcacccagg cgggctccca gcagccaccgccgcagcccc agcccccgcc 1200 gcctcctcca cccgcgtccc agcagccacc acccccgtcacccccacagg cgcccgtccg 1260 cctgccccct ggtggccccc tgttgcccag cgccagcctgactcgtgggc cacagccgcc 1320 cccgcttgca gtcacggtac cgtcctctct cccccagtcccccccagaga accctggcca 1380 gccatcgatg gggatcgaca tcgcctcggc gccggctctgcagcagtacc gcactagcgc 1440 cggctccccg gccaaccagt ctcccacctc gccagtctccaatcaaggct tctccccagg 1500 gagctccccg caactggagc agttcaacat gatggagaacgccatcagct ccagcagcct 1560 gtacagcccg ggctccacac tcaactactc gcaggcggccatgatgggcc tcacgggcag 1620 ccacgggagc ctgccggact cgcagcaact gggatacgccagccacggtg gcatccccaa 1680 catcatcctc acagtgacag gagagtcccc ccccagcctctctaaagaac tgaccagcca 1740 ccgaggacac cttccggatg gaccgcctgt gagcgggcacgccggcaccc tgccgctcag 1800 ccgtcccgac ggcgcctccc cagcccgggg acggccgtgctccgtccctc gccaacggcc 1860 gagcttgtga ttctgagctt gcaatgccgc caagcgccccccgccagccc gcccccggtt 1920 gtccacctcc cgcgaagccc aatcgcgagg ccgcgagccgggccgtccac ccacccgccc 1980 gcccagggct gggctgggat cggaggccgt gagcctcccgcccctgcaga ccctccctgc 2040 actggctccc tcgcccccag ccccggggcc tgagccgtcccctgtaagat gcgggaagtg 2100 tcagctcccg gcgtggcggg caggctcagg ggaggggcgcgcatggtccg ccagggctgt 2160 gggccgtggc gcattttccg actgtttgtc cagctctcactgccttcctt ggttcccggt 2220 cccccagccc atccgccatc cccagcccgt ggtcaggtagagagtgagcc ccacgccgcc 2280 ccagggagga ggcgccagag cgcggggcag acgcaaagtgaaataaacac tattttgacg 2340 gc 2342 5 31 PRT Homo sapiens 5 Tyr Glu ArgGly Arg Ala Glu Ser Ser Asp Arg Glu Arg Glu Ser Thr 1 5 10 15 Leu GlnLeu Leu Ser Leu Val Gln His Gly Gln Gly Ala Arg Lys 20 25 30 6 31 PRTHomo sapiens 6 Tyr Glu Ala Val Ser Pro Glu Arg Leu Glu Leu Glu Arg GlnHis Thr 1 5 10 15 Phe Ala Leu His Gln Arg Cys Ile Gln Ala Lys Ala LysArg Ala 20 25 30 7 31 PRT Homo sapiens 7 Tyr Gln Gln Ala Gln Val Glu GlnLeu Glu Leu Glu Arg Arg Asp Thr 1 5 10 15 Val Ser Leu Tyr Gln Arg ThrLeu Glu Gln Arg Ala Lys Lys Ser 20 25 30 8 31 PRT Drosophilamelanogaster 8 Tyr Glu Gln Ala Phe Asn Thr Val Cys Glu Gln Gln Asn GlnGlu Thr 1 5 10 15 Thr Val Leu Gln Lys Arg Phe Leu Glu Ser Lys Asn LysArg Ala 20 25 30 9 31 PRT Caenorhabditis elegans 9 Tyr Glu Lys Ala ArgPro Glu Met Ile Ala Asn Gln Arg Ala Val Thr 1 5 10 15 Ala His Leu PheAsn Arg Tyr Thr Glu Asp Glu Glu Arg Lys Arg 20 25 30 10 23 DNAArtificial Synthetic 10 cgagaagatg gcgacttcga aca 23 11 28 DNAArtificial Synthetic 11 ccattgggtc gcttgctgtt ggcaggag 28 12 1024 PRTHomo sapiens 12 Met Ala Thr Ser Asn Asn Pro Arg Lys Phe Ser Glu Lys IleAla Leu 1 5 10 15 His Asn Gln Lys Gln Ala Glu Glu Thr Ala Ala Phe GluGlu Val Met 20 25 30 Lys Asp Leu Ser Leu Thr Arg Ala Ala Arg Leu Gln GlySer Leu Lys 35 40 45 Arg Lys Gln Val Val Asn Leu Ser Pro Ala Asn Ser LysArg Pro Asn 50 55 60 Gly Phe Val Asp Asn Ser Phe Leu Asp Ile Lys Arg IleArg Val Gly 65 70 75 80 Glu Asn Leu Ser Ala Gly Gln Gly Gly Leu Gln IleAsn Asn Gly Gln 85 90 95 Ser Gln Ile Met Ser Gly Thr Leu Pro Met Ser GlnAla Pro Leu Arg 100 105 110 Lys Thr Asn Thr Leu Pro Ser His Thr His SerPro Gly Asn Gly Leu 115 120 125 Phe Asn Met Gly Leu Lys Glu Val Lys LysGlu Pro Gly Glu Thr Leu 130 135 140 Ser Cys Ser Lys His Met Asp Gly GlnMet Thr Gln Glu Asn Ile Phe 145 150 155 160 Pro Asn Arg Tyr Gly Asp AspPro Gly Glu Gln Leu Met Asp Pro Glu 165 170 175 Leu Gln Glu Leu Phe AsnGlu Leu Thr Asn Ile Ser Val Pro Pro Met 180 185 190 Ser Asp Leu Glu LeuGlu Asn Met Ile Asn Ala Thr Ile Lys Gln Asp 195 200 205 Asp Pro Phe AsnIle Asp Leu Gly Gln Gln Ser Gln Arg Ser Thr Pro 210 215 220 Arg Pro SerLeu Pro Met Glu Lys Ile Val Ile Lys Ser Glu Tyr Ser 225 230 235 240 ProGly Leu Thr Gln Gly Pro Ser Gly Ser Pro Gln Leu Arg Pro Pro 245 250 255Ser Ala Gly Pro Ala Phe Ser Met Ala Asn Ser Ala Leu Ser Thr Ser 260 265270 Ser Pro Ile Pro Ser Val Pro Gln Ser Gln Ala Gln Pro Gln Thr Gly 275280 285 Ser Gly Ala Ser Arg Ala Leu Pro Ser Trp Gln Glu Val Ser His Ala290 295 300 Gln Gln Leu Lys Gln Ile Ala Ala Asn Arg Gln Gln His Ala ArgMet 305 310 315 320 Gln Gln His Gln Gln Gln His Gln Pro Thr Asn Trp SerAla Leu Pro 325 330 335 Ser Ser Ala Gly Pro Ser Pro Gly Pro Phe Gly GlnGlu Lys Ile Pro 340 345 350 Ser Pro Ser Phe Gly Gln Gln Thr Phe Ser ProGln Ser Ser Pro Met 355 360 365 Pro Gly Val Ala Gly Gly Ser Gly Gln SerLys Val Met Ala Asn Tyr 370 375 380 Met Tyr Lys Ala Gly Pro Ser Ala GlnGly Gly His Leu Asp Val Leu 385 390 395 400 Met Gln Gln Lys Pro Gln AspLeu Ser Arg Ser Phe Ile Asn Asn Pro 405 410 415 His Pro Ala Met Glu ProArg Gln Gly Asn Thr Lys Pro Leu Phe His 420 425 430 Phe Asn Ser Asp GlnAla Asn Gln Gln Met Pro Ser Val Leu Pro Ser 435 440 445 Gln Asn Lys ProSer Leu Leu His Tyr Thr Gln Gln Gln Gln Gln Gln 450 455 460 Gln Gln GlnGln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 465 470 475 480 GlnGln Gln Gln Gln Gln Gln Gln Gln Ser Ser Ile Ser Ala Gln Gln 485 490 495Gln Gln Gln Gln Gln Ser Ser Ile Ser Ala Gln Gln Gln Gln Gln Gln 500 505510 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 515520 525 Gln Gln Gln Gln Gln Pro Ser Ser Gln Pro Ala Gln Ser Leu Pro Ser530 535 540 Gln Pro Leu Leu Arg Ser Pro Leu Pro Leu Gln Gln Lys Leu LeuLeu 545 550 555 560 Gln Gln Met Gln Asn Gln Pro Ile Ala Gly Met Gly TyrGln Val Ser 565 570 575 Gln Gln Gln Arg Gln Asp Gln His Ser Val Val GlyGln Asn Thr Gly 580 585 590 Pro Ser Pro Ser Pro Asn Pro Cys Ser Asn ProAsn Thr Gly Ser Gly 595 600 605 Tyr Met Asn Ser Gln Gln Ser Leu Leu AsnGln Gln Leu Met Gly Lys 610 615 620 Lys Gln Thr Leu Gln Arg Gln Ile MetGlu Gln Lys Gln Gln Leu Leu 625 630 635 640 Leu Gln Gln Gln Met Leu AlaAsp Ala Glu Lys Ile Ala Pro Gln Asp 645 650 655 Gln Ile Asn Arg His LeuSer Arg Pro Pro Pro Asp Tyr Lys Asp Gln 660 665 670 Arg Arg Asn Val GlyAsn Met Gln Pro Thr Ala Gln Tyr Ser Gly Gly 675 680 685 Ser Ser Thr IleSer Leu Asn Ser Asn Gln Ala Leu Ala Asn Pro Val 690 695 700 Ser Thr HisThr Ile Leu Thr Pro Asn Ser Ser Leu Leu Ser Thr Ser 705 710 715 720 HisGly Thr Arg Met Pro Ser Leu Ser Thr Ala Val Gln Asn Met Gly 725 730 735Met Tyr Gly Asn Leu Pro Cys Asn Gln Pro Asn Thr Tyr Ser Val Thr 740 745750 Ser Gly Met Asn Gln Leu Thr Gln Gln Arg Asn Pro Lys Gln Leu Leu 755760 765 Ala Asn Gln Asn Asn Pro Met Met Pro Arg Pro Pro Thr Leu Gly Pro770 775 780 Ser Asn Asn Asn Asn Val Ala Thr Phe Gly Ala Gly Ser Val GlyAsn 785 790 795 800 Ser Gln Gln Leu Arg Pro Asn Leu Thr His Ser Met AlaSer Met Pro 805 810 815 Pro Gln Arg Thr Ser Asn Val Met Ile Thr Ser AsnThr Thr Ala Pro 820 825 830 Asn Trp Ala Ser Gln Glu Gly Thr Ser Lys GlnGln Glu Ala Leu Thr 835 840 845 Ser Ala Gly Val Arg Phe Pro Thr Gly ThrPro Ala Ala Tyr Thr Pro 850 855 860 Asn Gln Ser Leu Gln Gln Ala Val GlySer Gln Gln Phe Ser Gln Arg 865 870 875 880 Ala Val Ala Pro Pro Asn GlnLeu Thr Pro Ala Val Gln Met Arg Pro 885 890 895 Met Asn Gln Met Ser GlnThr Leu Asn Gly Gln Thr Met Gly Pro Leu 900 905 910 Arg Gly Leu Asn LeuArg Pro Asn Gln Leu Ser Thr Gln Ile Leu Pro 915 920 925 Asn Leu Asn GlnSer Gly Thr Gly Leu Asn Gln Ser Arg Thr Gly Ile 930 935 940 Asn Gln ProPro Ser Leu Thr Pro Ser Asn Phe Pro Ser Pro Asn Gln 945 950 955 960 SerSer Arg Ala Phe Gln Gly Thr Asp His Ser Ser Asp Leu Ala Phe 965 970 975Asp Phe Leu Ser Gln Gln Asn Asp Asn Met Gly Pro Ala Leu Asn Ser 980 985990 Asp Ala Asp Phe Ile Asp Ser Leu Leu Lys Thr Glu Pro Gly Asn Asp 9951000 1005 Asp Trp Met Lys Asp Ile Asn Leu Asp Glu Ile Leu Gly Asn Asn1010 1015 1020 Ser

1. A method of screening a tissue sample from a subject for at(11;19)(q14-21;p12-13) translocation, comprising detecting the presenceof a MECT1-MAML2 chimeric nucleic acid in a tissue sample.
 2. The methodof claim 1, wherein said tissue sample comprises biopsy material.
 3. Themethod of claim 2, wherein said biopsy material comprises cells from asalivary gland tumor.
 4. The method of claim 3, wherein said salivarygland tumor is selected from the group consisting of a mucoepidermoidcancer, a pleomorphic adenoma, and a adenoid cystic carcinoma.
 5. Themethod of claim 1, wherein said MECT1-MAML2 chimeric nucleic acidcomprises DNA.
 6. The method of claim 5, wherein said detecting is byfluorescence in situ hybridization.
 7. The method of claim 5, whereinsaid detecting is by amplifying at least a portion of said MECT1-MAML2DNA by polymerase chain reaction.
 8. The method of claim 5, wherein saiddetecting is by Southern blot.
 9. The method of claim 1, wherein saidMECT1-MAML2 chimeric nucleic acid comprises RNA.
 10. The method of claim9, wherein said detecting is by amplifying at least a portion of aMECT1-MAML2 mRNA by reverse-transcriptase polymerase chain reaction. 11.The method of claim 9, wherein said detecting is by Northern blot. 12.The method of claim 9, wherein said detecting is by microarray.
 13. Amethod of screening a tissue sample from a subject for at(11;19)(q14-21;p12-13) translocation, comprising detecting the presenceof a MECT1-MAML2 chimeric protein in a tissue sample.
 14. The method ofclaim 13, wherein said detecting is by immunoblot.
 15. The method ofclaim 13, wherein said detecting is by immunofluorescence analysis. 16.A kit for screening a tissue sample from a subject for at(11;19)(q14-21;p12-13) translocation, comprising: a) a reagent capableof specifically detecting the presence of a MECT1-MAML2 chimeric nucleicacid in a tissue sample; and b) instructions for using said kit forscreening a tissue sample from a subject for a t(11;19)(q14-21;p12-13)translocation.
 17. The kit of claim 16, wherein said reagent comprises afirst nucleic acid probe complementary to at least a portion of MECT1exons 2-18, and a second nucleic acid probe complementary to at least aportion of MAML2 exon
 1. 18. The kit of claim 16, wherein said reagentcomprises a first nucleic acid probe complementary to at least a portionof MECT1 exon 1, and a second nucleic acid probe complementary to atleast a portion of MAML2 exons 2-5.
 19. The kit of claim 17, whereinsaid reagent comprises a first bacterial artificial chromosomedesignated as RP11-676L3, and a second bacterial artificial chromosomedesignated as RP11-16K5.
 20. The kit of claim 17, wherein said firstnucleic acid probe comprises a sense oligonucleotide, and said secondnucleic acid probe comprises an antisense oligonucleotide.
 21. A methodof screening compounds, comprising: a) providing: i) a cell containing aMECT1-MAML1 chimeric gene; and ii) at least one test compound; and b)contacting said cell with said test compound; and c) detecting a changein MECT1-MAML2 expression in said cell in the presence of said testcompound relative to the absence of said test compound.
 22. The methodof claim 21, wherein said cell is selected from the group consisting ofa cell transfected with a MECT1-MAML2 expression vector, and a cell witha t(11;19)(q14-21;p12-13) translocation.
 23. The method of claim 21,wherein said cell is selected from the group consisting of a cell invitro and a cell in vivo.
 24. The method of claim 21, wherein saiddetecting comprises detecting MECT1-MAML2 mRNA.
 25. The method of claim21, wherein said detecting comprises detecting MECT1-MAML2 protein.